Non-specific binding resistant protein arrays and methods for making the same

ABSTRACT

Arrays of protein-capture agents useful for the simultaneous detection of a plurality of proteins which are the expression products, or fragments thereof, of a cell or population of cells in an organism are provided. A variety of antibody arrays, in particular, are described. Methods of both making and using the arrays of protein-capture agents are also disclosed. The invention arrays are particularly useful for various proteomics applications including assessing patterns of protein expression and modification in cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/353,555 filed Jul. 14, 1999 (now U.S. Pat. No. 6,329,209)which is a continuation-in-part of U.S. patent application Ser. No.09/115,455 filed Jul. 14, 1998 (now U.S. Pat. No. 6,406,921) both herebyincorporated by reference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

a) Field of the Invention

The present invention relates generally to arrays of protein-captureagents and methods for the parallel detection and analysis of up to alarge number of proteins in a sample. More specifically, the presentinvention relates to proteomics and the measurement of gene activity atthe protein level in cells.

b) Description of Related Art

Although attempts to evaluate gene activity and to decipher biologicalprocesses including those of disease processes and drug effects havetraditionally focused on genomics, proteomics offers a more direct andpromising look at the biological functions of a cell. Proteomicsinvolves the qualitative and quantitative measurement of gene activityby detecting and quantitating expression at the protein level, ratherthan at the messenger RNA level. Proteomics also involves the study ofnon-genome encoded events including the post-translational modificationof proteins, interactions between proteins, and the location of proteinswithin the cell. The structure, function, or level of activity of theproteins expressed by a cell are also of interest. Essentially,proteomics involves the study of part or all of the status of the totalprotein contained within or secreted by a cell.

The study of gene expression at the protein level is important becausemany of the most important cellular processes are regulated by theprotein status of the cell, not by the status of gene expression. Also,the protein content of a cell is highly relevant to drug discoveryefforts since most drugs are designed to be active against proteintargets.

Measuring the mRNA abundances of a cell provides only an indirect andincomplete assessment of the protein content of a cell. The level ofactive protein that is produced in a cell is often determined by factorsother than the amount of mRNA produced. For instance, both proteinmaturation and protein degradation are actively controlled in the celland a protein's activity status can be regulated by post-translationalmodifications. Studies comparing mRNA transcript abundances to proteinabundances have found only a limited correlation (coefficient of about0.43-0.48) between the two (Anderson and Anderson, Electrophoresis,19:1853-1861, 1998). Furthermore, the extreme lability of RNA in samplesdue to chemical and enzymatic degradation makes the evaluation ofgenetic expression at the protein level more practical than at the mRNAlevel.

Current technologies for the analysis of proteomes are based on avariety of protein separation techniques followed by identification ofthe separated proteins. The most popular method is based on 2D-gelelectrophoresis followed by “in-gel” proteolytic digestion and massspectroscopy. Alternatively, Edman methods may be used for thesequencing. This 2D-gel technique requires large sample sizes, is timeconsuming, and is currently limited in its ability to reproduciblyresolve a significant fraction of the proteins expressed by a humancell. Techniques involving some large-format 2D-gels can produce gelswhich separate a larger number of proteins than traditional 2D-geltechniques, but reproducibility is still poor and over 95% of the spotscannot be sequenced due to limitations with respect to sensitivity ofthe available sequencing techniques. The electrophoretic techniques arealso plagued by a bias towards proteins of high abundance.

Standard assays for the presence of an analyte in a solution, such asthose commonly used for diagnostics, for instance, involve the use of anantibody which has been raised against the targeted antigen.Multianalyte assays known in the art involve the use of multipleantibodies and are directed towards assaying for multiple analytes.However, these multianalyte assays have not been directed towardsassaying the total or partial protein content of a cell or cellpopulation. Furthermore, sample sizes required to adapt such standardantibody assay approaches to the analysis of even a fraction of theestimated 100,000 or more different proteins of a human cell and theirvarious modified states are prohibitively large. Automation and/orminiaturization of antibody assays are required if large numbers ofproteins are to be assayed simultaneously. Materials, surface coatings,and detection methods used for macroscopic immunoassays and affinitypurification are not readily transferable to the formation orfabrication of miniaturized protein arrays.

Miniaturized DNA chip technologies have been developed (for example, seeU.S. Pat. Nos. 5,412,087, 5,445,934, and 5,744,305) and are currentlybeing exploited for the screening of gene expression at the mRNA level.These chips can be used to determine which genes are expressed bydifferent types of cells and in response to different conditions.However, DNA biochip technology is not transferable to protein-bindingassays such as antibody assays because the chemistries and materialsused for DNA biochips are not readily transferable to use with proteins.Nucleic acids such as DNA withstand temperatures up to 100° C., can bedried and re-hydrated without loss of activity, and can be boundphysically or chemically directly to organic adhesion layers supportedby materials such as glass while maintaining their activity. Incontrast, proteins such as antibodies are preferably kept hydrated andat ambient temperatures are sensitive to the physical and chemicalproperties of the support materials. Therefore, maintaining proteinactivity at the liquid-solid interface requires entirely differentimmobilization strategies than those used for nucleic acids. The properorientation of the antibody or other protein at the interface isdesirable to ensure accessibility of their active sites with interactingmolecules. With miniaturization of the chip and decreased feature sizes,the ratio of accessible to non-accessible and the ratio of active toinactive antibodies or proteins become increasingly relevant andimportant.

Thus, there is a need for the ability to assay in parallel a multitudeof proteins expressed by a cell or a population of cells in an organism,including up to the total set of proteins expressed by the cell orcells.

SUMMARY OF THE INVENTION

The present invention is directed to arrays of protein-capture agentsand methods of use thereof that satisfy the need to assay in parallel amultitude of proteins expressed by a cell or population of cells in anorganism, including up to the total protein content of a cell.

In one embodiment, the present invention provides an array ofprotein-capture agents comprising: a substrate; at least one organicthinfilm covering some or all of the surface of the substrate; and aplurality of patches arranged in discrete, known regions on the portionsof the substrate surface covered by organic thinfilm, wherein (i) eachpatch comprises protein-capture agents immobilized on the organicthinfilm, where the protein-capture agents of a given patch are capableof binding a particular expression product, or a fragment thereof, of acell or population of cells in an organism; and (ii) the array comprisesa plurality of different protein-capture agents, each of which iscapable of binding a different expression product, or fragment thereof,of the cell or population of cells in the organism.

In a second embodiment, the invention provides an array of boundproteins which comprises both the array of protein-capture agents of theinvention and a plurality of different proteins which are expressionproducts, or fragments thereof, of a cell or population of cells in anorganism, where each of the different proteins is bound to aprotein-capture agent on a separate patch of the array.

Methods of using the arrays of protein-capture agents of the inventionare also provided. In one embodiment of the invention, a method ofassaying in parallel for a plurality of different proteins in a samplewhich are expression products, or fragments thereof, of a cell or apopulation of cells in an organism, is provided which comprises firstdelivering the sample to the array of protein-capture agents of theinvention under conditions suitable for protein binding, wherein each ofthe proteins being assayed is a binding partner of the protein-captureagent of at least one patch on the array. The final step comprisesdetecting, either directly or indirectly, for the presence or amount ofprotein bound to each patch of the array. This method optionally furthercomprises the step of further characterizing the proteins bound to atleast one patch of the array.

In another embodiment of the invention, a method for determining theprotein expression pattern of a cell or a population of cells in anorganism is provided which comprises first delivering a samplecontaining the expression products, or fragments thereof, of the cell orpopulation of cells to the array of protein-capture agents of theinvention under conditions suitable for protein binding. The final stepcomprises detecting, either directly or indirectly, for the presence oramount of protein bound to each patch of the array. In an alternativeembodiment, a similar method for comparing the protein expressionpatterns of two cells or populations of cells is also provided.

In still another embodiment of the invention, an alternative method ofassaying in parallel for a plurality of different proteins in a samplewhich are expression products, or fragments thereof, of a cell or apopulation of cells in an organism is provided. The method of thisembodiment comprises first contacting the sample with an array ofspatially distinct patches of different protein-capture agents underconditions suitable for protein binding, wherein each of the proteinsbeing assayed is a binding partner of the protein-capture agent of atleast one patch on the array. The last step of the method involvesdetecting, either directly or indirectly, for the presence or amount ofprotein bound to each patch of the array.

In a still further embodiment, a method of producing an array ofprotein-capture agents is provided which comprises the following steps:selecting protein-capture agents from a library of protein-captureagents, wherein the protein-capture agents are selected by their bindingaffinity to the proteins from a cellular extract or body fluid;producing a plurality of purified samples of the selectedprotein-capture agents; and immobilizing the protein-capture agent ofeach different purified sample onto an organic thinfilm on a separatepatch on the substrate surface to form a plurality of patches ofprotein-capture agents on discrete, known regions of the surface of asubstrate.

In an alternative embodiment, the invention provides a method forproducing an array of protein-capture agents which comprises a firststep of selecting protein-capture agents from a library ofprotein-capture agents, wherein the protein-capture agents are selectedby their binding affinity to proteins which are the expression products,or fragments thereof, of a cDNA expression library. The second step ofthe method comprises producing a plurality of purified samples of theprotein-capture agents selected in the first step. The third stepcomprises immobilizing the protein-capture agent of each differentpurified sample onto an organic thinfilm on a separate patch on thesubstrate surface to form a plurality of patches of protein-captureagents on discrete, known regions of the surface of a substrate.

In another aspect, the invention provides for array devices comprising asubstrate having a surface; and, one or more protein immobilizationregions on said surface, said protein immobilization regions eachcomprising; an ordered hydrophobic monolayer formed of alkyl chainshaving proximal ends which are chemisorbed or physisorbed to saidsurface within said immobilization regions, and opposite hydrophobicdistal ends; a hydrophilic monolayer attached to said orderedhydrophobic monolayer, said hydrophilic monolayer comprising a set offirst hydrophilic chains, each first hydrophilic chain having a proximalend by which said first hydrophilic chain is linked to an alkyl chaindistal end, and an opposite hydrophilic distal end having a functionalgroup for covalently attaching a protein capture agent thereto; one ormore of said protein capture agents attached to a first subset of saidset of first hydrophilic chains within said immobilization regionsthrough a residue formed by reacting said protein capture agents withsaid functional groups of said first subset of said first hydrophilicchains; and, one or more second hydrophilic chains each being attachedto a second subset of said set of said first hydrophilic chains throughsaid functional group or residue thereof, wherein said second subset offirst hydrophilic chains and said first subset of first hydrophilicchains are mutually exclusive of each other; wherein said firsthydrophilic chains and said second hydrophilic chains are effective toresist non-specific protein binding. In preferred embodiments, thedevice has said second hydrophilic chains being polyethylene glycolchains and/or said first hydrophilic chains being oligoethylene glycolchains and/or at least two of said protein immobilization regions beingadjacent each other and/or at least two of said protein immobilizationregions are separated from each other by one or more border regions.

In another aspect, the invention provides for a method for making anarray device comprising the steps of: providing a substrate having asurface; and one or more protein immobilization regions on said surface,said protein immobilization regions each comprising; an orderedhydrophobic monolayer formed of alkyl chains having proximal ends whichare chemisorbed or physisorbed to said surface within saidimmobilization regions, and opposite hydrophobic distal ends; ahydrophilic monolayer attached to said ordered hydrophobic monolayer,said hydrophilic monolayer comprising a set of first hydrophilic chains,each first hydrophilic chain having a proximal end by which said firsthydrophilic chain is linked to an alkyl chain distal end, and anopposite hydrophilic distal end; one or more functional groups, each forcovalently attaching a protein capture agent thereto and each covalentlyattached to one or more of said hydrophilic distal ends of said firsthydrophilic chains; reacting one or more of said protein capture agentsto one or more of said functional groups attached to a first subset ofsaid set of first hydrophilic chains within said immobilization regionsto attach said one or more protein capture agents through a residueformed by said reaction of said protein capture agents with saidfunctional groups of said first subset of said first hydrophilic chains;and, reacting one or more second hydrophilic chains with a second subsetof said set of first hydrophilic chains so that one or more of saidsecond hydrophilic chains attach through one or more residues of one ormore of said second subset of said first hydrophilic chains. Inpreferred embodiments, the method has at least one of said secondhydrophilic chains being polyethylene glycol and/or at least one of saidfirst hydrophilic chains being oligoethylene glycol.

A method for making an array device comprising the steps of: providing asubstrate having a surface; and one or more protein immobilizationregions on said surface, said protein immobilization regions eachcomprising; an ordered hydrophobic monolayer formed from a set of alkylchains having proximal ends which are chemisorbed or physisorbed to saidsurface within said immobilization regions, and opposite hydrophobicdistal ends; one or more functional groups for covalently attaching saidprotein captures agent thereto, each functional group being attached tosaid hydrophobic distal ends of a subset of said set of alkyl chains;reacting one or more of said protein capture agents to one or more ofsaid functional groups attached to a first subset of said set of alkylchains within said immobilization regions to attach said one or moreprotein capture agents through a residue formed by said reaction of saidprotein capture agents with said functional groups of said first subsetof said alkyl chains; and, reacting one or more hydrophilic chains witha second subset of said set of alkyl chains so that one or more of saidsecond hydrophilic chains attach through one or more residues of one ormore of said second subset of said set of alkyl chains, wherein saidfirst subset of said set of alkyl chains and said second subset of saidset of alkyl chains are mutually exclusive. In preferred embodiments, atleast one of said hydrophilic chains is polyethylene glycol. and/or atleast one of said first hydrophilic chains is oligoethylene glycol.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the top view of an array of patches reactive towardsprotein-capture agents.

FIG. 2 shows the cross section of an individual patch of the array ofFIG. 1.

FIG. 3 shows the cross section of a row of monolayer-covered patches ofthe array of FIG. 1.

FIG. 4 shows a thiolreactive monolayer on a substrate.

FIG. 5 shows an aminoreactive monolayer on a coated substrate.

FIG. 6 shows the immobilization of a protein-capture agent on amonolayer-coated substrate via an affinity tag.

FIG. 7 shows the immobilization of a protein-capture agent on amonolayer-coated substrate via an affinity tag and an adaptor.

FIG. 8 shows a schematic of a fluorescence detection unit which may beused to monitor binding of proteins by the protein-capture agents of thearray.

FIG. 9 shows a schematic of an ellipsometric detection unit which may beused to monitor binding of proteins by the protein-capture agents of thearray.

FIGS. 10A-C show several panels where unreacted functional groups arequenched.

DETAILED DESCRIPTION OF THE INVENTION

A variety of arrays of protein-capture agents and methods useful formultianalyte analyses and analyses of protein expression andmodification in cells are provided by the present invention.

(a) Definitions

The term “protein-capture agent” means a molecule or a multi-molecularcomplex which can bind a protein to itself. Protein-capture agentspreferably bind their binding partners in a substantially specificmanner. Protein-capture agents with a dissociation constant (K_(D)) ofless than about 10⁻⁶ are preferred. The protein-capture agent will mosttypically be a biomolecule such as a protein or a polynucleotide. Thebiomolecule may optionally be a naturally occurring, recombinant, orsynthetic biomolecule. Antibodies or antibody fragments are highlysuitable as protein-capture agents. Antigens may also serve asprotein-capture agents, since they are capable of binding antibodies. Areceptor which binds a protein ligand is another example of a possibleprotein-capture agent. For instance, protein-capture agents areunderstood not to be limited to agents which only interact with theirbinding partners through noncovalent interactions. Protein-captureagents may also optionally become covalently attached to proteins whichthey bind. For instance, the protein-capture agent may bephotocrosslinked to its binding partner following binding.

The term “binding partner” means a protein which is bound by aparticular protein-capture agent, preferably in a substantially specificmanner. In some cases, the protein-capture agent may be a cellular orextracellular protein and the binding partner may be the entity normallybound in vivo. In other embodiments, however, the binding partner may bethe protein or peptide on which the protein-capture agent was selected(through in vitro or in vivo selection) or raised (as in the case ofantibodies). A binding partner may be shared by more than oneprotein-capture agent. For instance, a binding partner which is bound bya variety of polyclonal antibodies may bear a number of differentepitopes. One protein-capture agent may also bind to a multitude ofbinding partners, for instance, if the binding partners share the sameepitope.

A “protein” means a polymer of amino acid residues linked together bypeptide bonds. The term, as used herein, refers to proteins,polypeptides, and peptides of any size, structure, or function.Typically, however, a protein will be at least six amino acids long.Preferably, if the protein is a short peptide, it will be at least about10 amino acid residues long. A protein may be naturally occurring,recombinant, or synthetic, or any combination of these. A protein mayalso be just a fragment of a naturally occurring protein or peptide. Aprotein may be a single molecule or may be a multi-molecular complex.The term protein may also apply to amino acid polymers in which one ormore amino acid residues is an artificial chemical analogue of acorresponding naturally occurring amino acid. An amino acid polymer inwhich one or more amino acid residues is an “unnatural” amino acid, notcorresponding to any naturally occurring amino acid, is also encompassedby the use of the term “protein” herein.

A “fragment of a protein” means a protein which is a portion of anotherprotein. For instance, fragments of a proteins may be a polypeptidesobtained by digesting full-length protein isolated from cultured cells.A fragment of a protein will typically comprise at least six aminoacids. More typically, the fragment will comprise at least ten aminoacids. Preferably, the fragment comprises at least about 16 amino acids.

An “expression product” is a biomolecule, such as a protein, which isproduced when a gene in an organism is expressed. An expression productmay optionally comprise post-translational modifications.

The term “antibody” means an immunoglobulin, whether natural orpartially or wholly synthetically produced. All derivatives thereofwhich maintain specific binding ability are also included in the term.The term also covers any protein having a binding domain which ishomologous or largely homologous to an immunoglobulin binding domain.These proteins may be derived from natural sources, or partly or whollysynthetically produced. An antibody may be monoclonal or polyclonal. Theantibody may be a member of any immunoglobulin class, including any ofthe human classes: IgG, IgM, IgA, IgD, and IgE. Derivatives of the IgGclass, however, are preferred in the present invention.

The term “antibody fragment” refers to any derivative of an antibodywhich is less than full-length. Preferably, the antibody fragmentretains at least a significant portion of the full-length antibody'sspecific binding ability. Examples of antibody fragments include, butare not limited to, Fab, Fab′, F(ab′)₂, scFv, Fv, dsFv diabody, and Fdfragments. The antibody fragment may be produced by any means. Forinstance, the antibody fragment may be enzymatically or chemicallyproduced by fragmentation of an intact antibody or it may berecombinantly produced from a gene encoding the partial antibodysequence. Alternatively, the antibody fragment may be wholly orpartially synthetically produced. The antibody fragment may optionallybe a single chain antibody fragment. Alternatively, the fragment maycomprise multiple chains which are linked together, for instance, bydisulfide linkages. The fragment may also optionally be a multimolecularcomplex. A functional antibody fragment will typically comprise at leastabout 50 amino acids and more typically will comprise at least about 200amino acids.

Single-chain Fvs (scFvs) are recombinant antibody fragments consistingof only the variable light chain (V_(L)) and variable heavy chain(V_(H)) covalently connected to one another by a polypeptide linker.Either V_(L) or V_(H) may be the NH₂-terminal domain. The polypeptidelinker may be of variable length and composition so long as the twovariable domains are bridged without serious steric interference.Typically, the linkers are comprised primarily of stretches of glycineand serine residues with some glutamic acid or lysine residuesinterspersed for solubility.

“Diabodies” are dimeric scFvs. The components of diabodies typicallyhave shorter peptide linkers than most scFvs and they show a preferencefor associating as dimers.

An “Fv” fragment consists of one V_(H) and one V_(L) domain heldtogether by noncovalent interactions. The term “dsFv” is used herein torefer to an Fv with an engineered intermolecular disulfide bond tostabilize the V_(H)-V_(L) pair.

A “F(ab′)₂” fragment is an antibody fragment essentially equivalent tothat obtained from immunoglobulins (typically IgG) by digestion with anenzyme pepsin at pH 4.0-4.5. The fragment may be recombinantly produced.

A “Fab′” fragment is an antibody fragment essentially equivalent to thatobtained by reduction of the disulfide bridge or bridges joining the twoheavy chain pieces in the F(ab′)₂ fragment. The Fab′ fragment may berecombinantly produced.

A “Fab” fragment is an antibody fragment essentially equivalent to thatobtained by digestion of immunoglobulins (typically IgG) with the enzymepapain. The Fab fragment may be recombinantly produced. The heavy chainsegment of the Fab fragment is the Fd piece.

A “population of cells in an organism” means a collection of more thanone cell in a single organism or more than one cell originally derivedfrom a single organism. The cells in the collection are preferably allof the same type. They may all be from the same tissue in an organism,for instance. Most preferably, gene expression in all of the cells inthe population is identical or nearly identical.

“Conditions suitable for protein binding” means those conditions (interms of salt concentration, pH, detergent, protein concentration,temperature, etc.) which allow for binding to occur between animmobilized protein-capture agent and its binding partner in solution.Preferably, the conditions are not so lenient that a significant amountof nonspecific protein binding occurs.

A “body fluid” may be any liquid substance extracted, excreted, orsecreted from an organism or tissue of an organism. The body fluid neednot necessarily contain cells. Body fluids of relevance to the presentinvention include, but are not limited to, whole blood, serum, urine,plasma, cerebral spinal fluid, tears, sinovial fluid, and amnioticfluid.

An “array” is an arrangement of entities in a pattern on a substrate.Although the pattern is typically a two-dimensional pattern, the patternmay also be a three-dimensional pattern.

A “patch of protein-capture agents” means a discrete region ofimmobilized protein-capture agents on the surface of a substrate. Thepatches may be of any geometric shape or may be irregularly shaped. Forinstance, the patch may be, but need not necessarily be, square inshape.

“Proteomics” means the study of or the characterization of either theproteome or some fraction of the proteome. The “proteome” is the totalcollection of the intracellular proteins of a cell or population ofcells and the proteins secreted by the cell or population of cells. Thischaracterization most typically includes measurements of the presence,and usually quantity, of the proteins which have been expressed by acell. The function, structural characteristics (such as posttranslational modification), and location within the cell of theproteins may also be studied. “Functional proteomics” refers to thestudy of the functional characteristics, activity level, and structuralcharacteristics of the protein expression products of a cell orpopulation of cells.

The term “substrate” refers to the bulk, underlying, and core materialof the arrays of the invention.

The terms “micromachining” and “microfabrication” both refer to anynumber of techniques which are useful in the generation ofmicrostructures (structures with feature sizes of sub-millimeter scale).Such technologies include, but are not limited to, laser ablation,electrodeposition, physical and chemical vapor deposition,photolithography, and wet chemical and dry etching. Related technologiessuch as injection molding and LIGA (X-ray lithography,electrodeposition, and molding) are also included. Most of thesetechniques were originally developed for use in semiconductors,microelectronics, and Micro-ElectroMechanical Systems (MEMS) but areapplicable to the present invention as well.

The term “coating” means a layer that is either naturally orsynthetically formed on or applied to the surface of the substrate. Forinstance, exposure of a substrate, such as silicon, to air results inoxidation of the exposed surface. In the case of a substrate made ofsilicon, a silicon oxide coating is formed on the surface upon exposureto air. In other instances, the coating is not derived from thesubstrate and may be placed upon the surface via mechanical, physical,electrical, or chemical means. An example of this type of coating wouldbe a metal coating that is applied to a silicon or polymer substrate ora silicon nitride coating that is applied to a silicon substrate.Although a coating may be of any thickness, typically the coating has athickness smaller than that of the substrate.

An “interlayer” is an additional coating or layer that is positionedbetween the first coating and the substrate. Multiple interlayers mayoptionally be used together. The primary purpose of a typical interlayeris to aid adhesion between the first coating and the substrate. One suchexample is the use of a titanium or chromium interlayer to help adhere agold coating to a silicon or glass surface. However, other possiblefunctions of an interlayer are also anticipated. For instance, someinterlayers may perform a role in the detection system of the array(such as a semiconductor or metal layer between a nonconductivesubstrate and a nonconductive coating).

An “organic thinfilm” is a thin layer of organic molecules which hasbeen applied to a substrate or to a coating on a substrate if present.Typically, an organic thinfilm is less than about 20 nm thick.Optionally, an organic thinfilm may be less than about 10 nm thick. Anorganic thinfilm may be disordered or ordered. For instance, an organicthinfilm can be amorphous (such as a chemisorbed or spin-coated polymer)or highly organized (such as a Langmuir-Blodgett film or self-assembledmonolayer). An organic thinfilm may be heterogeneous or homogeneous.Organic thinfilms which are monolayers are preferred. A lipid bilayer ormonolayer is a preferred organic thinfilm. Optionally, the organicthinfilm may comprise a combination of more than one form of organicthinfilm. For instance, an organic thinfilm may comprise a lipid bilayeron top of a self-assembled monolayer. A hydrogel may also compose anorganic thinfilm. The organic thinfilm will typically havefunctionalities exposed on its surface which serve to enhance thesurface conditions of a substrate or the coating on a substrate in anyof a number of ways. For instance, exposed functionalities of theorganic thinfilm are typically useful in the binding or covalentimmobilization of the protein-capture agents to the patches of thearray. Alternatively, the organic thinfilm may bear functional groups(such as polyethylene glycol (PEG)) which reduce the non-specificbinding of molecules to the surface. Other exposed functionalities serveto tehter the thinfilm to the surface of the substrate or the coating.Particular functionalities of the organic thinfilm may also be designedto enable certain detection techniques to be used with the surface.Alternatively, the organic thinfilm may serve the purpose of preventinginactivation of a protein-capture agent or the protein to be bound by aprotein-capture agent from occurring upon contact with the surface of asubstrate or a coating on the surface of a substrate.

A “monolayer” is a single-molecule thick organic thinfilm. A monolayermay be disordered or ordered. A monolayer may optionally be a polymericcompound, such as a polynonionic polymer, a polyionic polymer, or ablock-copolymer. For instance, the monolayer may be composed of apoly(amino acid) such as polylysine. A monolayer which is aself-assembled monolayer, however, is most preferred. One face of theself-assembled monolayer is typically composed of chemicalfunctionalities on the termini of the organic molecules that arechemisorbed or physisorbed onto the surface of the substrate or, ifpresent, the coating on the substrate if present. Examples of suitablefunctionalities of monolayers include the positively charged aminogroups of poly-L-lysine for use on negatively charged surfaces andthiols for use on gold surfaces. Typically, the other face of theself-assembled monolayer is exposed and may bear any number of chemicalfunctionalities (end groups). Preferably, the molecules of theself-assembled monolayer are highly ordered.

A “self-assembled monolayer” is a monolayer which is created by thespontaneous assembly of molecules. The self-assembled monolayer may beordered, disordered, or exhibit short- to long-range order.

An “affinity tag” is a functional moiety capable of directly orindirectly immobilizing a protein-capture agent onto an exposedfunctionality of the organic thinfilm. Preferably, the affinity tagenables the site-specific immobilization and thus enhances orientationof the protein-capture agent onto the organic thinfilm. In some cases,the affinity tag may be a simple chemical functional group. Otherpossibilities include amino acids, poly(amino acid) tags, or full-lengthproteins. Still other possibilities include carbohydrates and nucleicacids. For instance, the affinity tag may be a polynucleotide whichhybridizes to another polynucleotide serving as a functional group onthe organic thinfilm or another polynucleotide serving as an adaptor.The affinity tag may also be a synthetic chemical moiety. If the organicthinfilm of each of the patches comprises a lipid bilayer or monolayer,then a membrane anchor is a suitable affinity tag. The affinity tag maybe covalently or noncovalently attached to the protein-capture agent.For instance, if the affinity tag is covalently attached to theprotein-capture agent it may be attached via chemical conjugation or asa fusion protein. The affinity tag may also be attached to theprotein-capture agent via a cleavable linkage. Alternatively, theaffinity tag may not be directly in contact with the protein-captureagent. The affinity tag may instead be separated from theprotein-capture agent by an adaptor. The affinity tag may immobilize theprotein-capture agent to the organic thinfilm either through noncovalentinteractions or through a covalent linkage.

An “adaptor”, for purposes of this invention, is any entity that linksan affinity tag to the protein-capture agent. The adaptor may be, butneed not necessarily be, a discrete molecule that is noncovalentlyattached to both the affinity tag and the protein-capture agent. Theadaptor can instead be covalently attached to the affinity tag or theprotein-capture agent or both (via chemical conjugation or as a fusionprotein, for instance). Proteins such as full-length proteins,polypeptides, or peptides are typical adaptors. Other possible adaptorsinclude carbohydrates or nucleic acids.

The term “fusion protein” refers to a protein composed of two or morepolypeptides that, although typically unjoined in their native state,are joined by their respective amino and carboxyl termini through apeptide linkage to form a single continuous polypeptide. It isunderstood that the two or more polypeptide components can either bedirectly joined or indirectly joined through a peptide linker/spacer.

The term “normal physiological condition” means conditions that aretypical inside a living organism or a cell. While it is recognized thatsome organs or organisms provide extreme conditions, theintra-organismal and intra-cellular environment normally varies aroundpH 7 (i.e., from pH 6.5 to pH 7.5), contains water as the predominantsolvent, and exists at a temperature above 0° C. and below 50° C. Itwill be recognized that the concentration of various salts depends onthe organ, organism, cell, or cellular compartment used as a reference.

(b) Arrays of the Invention

The present invention is directed to arrays of protein-capture agentswhich can bind a plurality of proteins that are the expression products,or fragments thereof, of a cell or population of cells in an organismand therefore can be used to evaluate gene expression at the proteinlevel. Typically, the arrays comprise micrometer-scale, two-dimensionalpatterns of patches of protein-capture agents immobilized on an organicthinfilm coating on the surface of the substrate.

In one embodiment of the invention, the array of protein-capture agentscomprises a substrate, at least one organic thinfilm covering some orall of the surface of the substrate, and a plurality of patches arrangedin discrete, known regions on the portions of the substrate surfacecovered by organic thinfilm, wherein (i) each patch comprisesprotein-capture agents immobilized on the organic thinfilm, wherein saidprotein-capture agents of a given patch are capable of binding aparticular expression product, or a fragment thereof, of a cell orpopulation of cells in an organism, and (ii) the array comprises aplurality of different protein-capture agents, each of which is capableof binding a different expression product, or fragment thereof, of thecell or population of cells.

The protein-capture agents are preferably covalently immobilized on thepatches of the array, either directly or indirectly.

In most cases, the array will comprise at least about ten patches. In apreferred embodiment, the array comprises at least about 50 patches. Ina particularly preferred embodiment the array comprises at least about100 patches. In alternative preferred embodiments, the array ofprotein-capture agents may comprise more than 10³, 10⁴ or 10⁵ patches.

The area of surface of the substrate covered by each of the patches ispreferably no more than about 0.25 mm². Preferably, the area of thesubstrate surface covered by each of the patches is between about 1 μm²and about 10,000 μm². In a particularly preferred embodiment, each patchcovers an area of the substrate surface from about 100 μm² to about2,500 μm². In an alternative embodiment, a patch on the array may coveran area of the substrate surface as small as about 2,500 nm², althoughpatches of such small size are generally not necessary for the use ofthe array.

The patches of the array may be of any geometric shape. For instance,the patches may be rectangular or circular. The patches of the array mayalso be irregularly shaped. The patches are optionally elevated from themedian plan of the underlying substrate.

The distance separating the patches of the array can vary. Preferably,the patches of the array are separated from neighboring patches by about1 μm to about 500 μm. Typically, the distance separating the patches isroughly proportional to the diameter or side length of the patches onthe array if the patches have dimensions greater than about 10 μm. Ifthe patch size is smaller, then the distance separating the patches willtypically be larger than the dimensions of the patch.

In some versions of the array, the diameter of each of the patches isproportional to the distance separating the patches. Therefore, the areaof each patch may be from about 100 nm² to about 40,000 μm². Each patchpreferably has an area from about 1 μm² to about 10,000 μm².

In a preferred embodiment of the array, the patches of the array are allcontained within an area of about 1 cm² or less on the surface of thesubstrate. In one preferred embodiment of the array, therefore, thearray comprises 100 or more patches within a total area of about 1 cm²or less on the surface of the substrate. Alternatively, a particularlypreferred array comprises 10³ or more patches within a total area ofabout 1 cm² or less. A preferred array may even optionally comprise 10⁴or 10⁵ or more patches within an area of about 1 cm² or less on thesurface of the substrate. In other embodiments of the invention, all ofthe patches of the array are contained within an area of about 1 mm² orless on the surface of the substrate.

Typically, only one type of protein-capture agent is present on a singlepatch of the array. If more than one type of protein-capture agent ispresent on a single patch, all of the protein-capture agents of thatpatch must share a common binding partner. For instance, a patch maycomprise a variety of polyclonal antibodies to the same antigen(although, potentially, the antibodies may bind different epitopes onthat same antigen).

The arrays of the invention can have any number of a plurality ofdifferent protein-capture agents. Typically the array comprises at leastabout ten different protein-capture agents. Preferably, the arraycomprises at least about 50 different protein-capture agents. Morepreferably, the array comprises at least about 100 differentprotein-capture agents. Alternative preferred arrays comprise more thanabout 10³ different protein-capture agents or more than about 10⁴different protein-capture agents. The array may even optionally comprisemore than about 10⁵ different protein-capture agents.

The number of different protein-capture agents on the array will varydepending on the application desired. For instance, if the array is tobe used as a diagnostic tool in evaluating the status of a tumor orother diseased tissue in a patient, an array comprising less than about100 different protein-capture agents may suffice since the necessarybinding partners of the protein-capture agent on the array are limitedto only those proteins whose expression is known to be indicative of thedisease condition. However, if the array is to be used to measure asignificant portion of the total protein content of a cell, then thearray preferably comprises at least about 10,000 differentprotein-capture agents. Alternatively, a more limited proteomics study,such as a study of the abundances of various human transcriptionfactors, for instance, might only require an array of about 100different protein-capture agents.

In one embodiment of the array, each of the patches of the arraycomprises a different protein-capture agent. For instance, an arraycomprising about 100 patches could comprise about 100 differentprotein-capture agents. Likewise, an array of about 10,000 patches couldcomprise about 10,000 different protein-capture agents. In analternative embodiment, however, each different protein-capture agent isimmobilized on more than one separate patch on the array. For instance,each different protein-capture agent may optionally be present on two tosix different patches. An array of the invention, therefore, maycomprise about three-thousand protein-capture agent patches, but onlycomprise about one thousand different protein-capture agents since eachdifferent protein-capture agent is present on three different patches.

Typically, the number of different proteins which can be bound by theplurality of different protein-capture agents on the array will be atleast about ten. However, it is preferred that the plurality ofdifferent protein-capture agents on the array is capable of binding ahigher number of different proteins, such as at least about 50 or atleast about 100. In still further preferred embodiments, the pluralityof different proteins on the array is capable of binding at least about10³ proteins. For some applications, such as those where it is desirableto assay the entire protein content of a cell, or a significant fractionthereof, an array where the plurality of protein-capture agents iscapable of binding at least about 10⁴ different proteins or even atleast about 10⁵ different proteins is most preferred.

In one embodiment of the invention, the binding partners of theplurality of protein-capture agents on the array are proteins which areall expression products, or fragments thereof, of a cell or populationof cells of a single organism. The expression products may be proteins,including peptides, of any size or function. They may be intracellularproteins or extracellular proteins. The expression products may be froma one-celled or multicellular organism. The organism may be a plant oran animal. In a preferred embodiment of the invention, the bindingpartners are human expression products, or fragments thereof.

In one embodiment of the invention, the binding partners of theprotein-capture agents of the array may be a randomly chosen subset ofall the proteins, including peptides, which are expressed by a cell orpopulation of cells in a given organism or a subset of all the fragmentsof those proteins. Thus, the binding partners of the protein-captureagents of the array optionally represent a wide distribution ofdifferent proteins from a single organism.

The binding partners of some or all of the protein-capture agents on thearray need not necessarily be known. The binding partner of aprotein-capture agent of the array may be a protein or peptide ofunknown function. For instance, the different protein-capture agents ofthe array may together bind a wide range of cellular proteins from asingle cell type, many of which are of unknown identity and/or function.

In another embodiment of the present invention, the binding partners ofthe protein-capture agents on the array are related proteins. Thedifferent proteins bound by the protein-capture agents may optionally bemembers of the same protein family. The different binding partners ofthe protein-capture agents of the array may be either functionallyrelated or just suspected of being functionally related. The differentproteins bound by the protein-capture agents of the array may also beproteins which share a similarity in structure or sequence or are simplysuspected of sharing a similarity in structure or sequence. Forinstance, the binding partners of the protein-capture agents on thearray may optionally all be growth factor receptors, hormone receptors,neurotransmitter receptors, catecholamine receptors, amino acidderivative receptors, cytokine receptors, extracellular matrixreceptors, antibodies, lectins, cytokines, serpins, proteases, kinases,phosphatases, ras-like GTPases, hydrolases, steroid hormone receptors,transcription factors, heat-shock transcription factors, DNA-bindingproteins, zinc-finger proteins, leucine-zipper proteins, homeodomainproteins, intracellular signal transduction modulators and effectors,apoptosis-related factors, DNA synthesis factors, DNA repair factors,DNA recombination factors, cell-surface antigens, hepatitis C virus(HCV) proteases or HIV proteases.

In an alternative embodiment of the invention, the proteins which arethe binding partners of the protein-capture agents of the array may befragments of the expression products of a cell or population of cells inan organism.

A protein-capture agent on the array can be any molecule or complex ofmolecules which has the ability to bind a protein and immobilize it tothe site of the protein-capture agent on the array. Preferably, theprotein-capture agent binds its binding partner in a substantiallyspecific manner. Hence, the protein-capture agent may optionally be aprotein whose natural function in a cell is to specifically bind anotherprotein, such as an antibody or a receptor. Alternatively, theprotein-capture agent may instead be a partially or wholly synthetic orrecombinant protein which specifically binds a protein. Alternatively,the protein-capture agent may be a protein which has been selected invitro from a mutagenized, randomized, or completely random and syntheticlibrary by its binding affinity to a specific protein or peptide target.The selection method used may optionally have been a display method suchas ribosome display or phage display (see below). Alternatively, theprotein-capture agent obtained via in vitro selection may be a DNA orRNA aptamer which specifically binds a protein target (for example:Potyrailo et al., Anal. Chem., 70:3419-25, 1998; Cohen, et al., Proc.Natl. Acad. Sci. USA, 95:14272-7, 1998; Fukuda, et al., Nucleic AcidsSymp. Ser., (37):237-8, 1997). Alternatively, the in vitro selectedprotein-capture agent may be a polypeptide (Roberts and Szostak, Proc.Natl. Acad. Sci. USA, 94:12297-302, 1997). In an alternative embodiment,the protein-capture agent may be a small molecule which has beenselected from a combinatorial chemistry library or is isolated from anorganism.

In a preferred embodiment of the array, however, the protein-captureagents are proteins. In a particularly preferred embodiment, theprotein-capture agents are antibodies or antibody fragments. Althoughantibody moieties are exemplified herein, it is understood that thepresent arrays and methods may be advantageously employed with otherprotein-capture agents.

The antibodies or antibody fragments of the array may optionally besingle-chain Fvs, Fab fragments, Fab′ fragments, F(ab′)₂ fragments, Fvfragments, dsFvs diabodies, Fd fragments, full-length, antigen-specificpolyclonal antibodies, or full-length monoclonal antibodies. In apreferred embodiment, the protein-capture agents of the array aremonoclonal antibodies, Fab fragments or single-chain Fvs.

The antibodies or antibody fragments may be monoclonal antibodies, evencommercially available antibodies, against known, well-characterizedproteins. Alternatively, the antibody fragments have been derived byselection from a library using the phage display method. If the antibodyfragments are derived individually by selection based on bindingaffinity to known proteins, then, the binding partners of the antibodyfragments are known. In an alternative embodiment of the invention, theantibody fragments have been derived by a phage display methodcomprising selection based on binding affinity to the (typically,immobilized) proteins of a cellular extract or a body fluid. In thisembodiment, some or many of the antibody fragments of the array wouldbind proteins of unknown identity and/or function.

Upon using the array of protein-capture agents to bind a plurality ofexpression products, or fragments thereof, an array of bound proteins iscreated. Thus, another embodiment of the invention provides an array ofbound proteins which comprises (a) a protein-capture agent array of theinvention and (b) a plurality of different proteins which are expressionproducts, or fragments thereof, of a cell or a population of cells in anorganism, wherein each of the different proteins is bound to aprotein-capture agent on a separate patch of the array. Preferably, eachof the different proteins is non-covalently bound to a protein-captureagent.

(c) Substrates, Coatings, and Organic Thinfilms

The substrate of the array may be either organic or inorganic,biological or non-biological, or any combination of these materials. Inone embodiment, the substrate is transparent or translucent. The portionof the surface of the substrate on which the patches reside ispreferably flat and firm or semi-firm. However, the array of the presentinvention need not necessarily be flat or entirely two-dimensional.Significant topological features may be present on the surface of thesubstrate surrounding the patches, between the patches or beneath thepatches. For instance, walls or other barriers may separate the patchesof the array.

Numerous materials are suitable for use as a substrate in the arrayembodiment of the invention. For instance, the substrate of theinvention array can comprise a material selected from a group consistingof silicon, silica, quartz, glass, controlled pore glass, carbon,alumina, titania, tantalum oxide, germanium, silicon nitride, zeolites,and gallium arsenide. Many metals such as gold, platinum, aluminum,copper, titanium, and their alloys are also options for substrates ofthe array. In addition, many ceramics and polymers may also be used assubstrates. Polymers which may be used as substrates include, but arenot limited to, the following: polystyrene; poly(tetra)fluoroethylene(PTFE); polyvinylidenedifluoride; polycarbonate; polymethylmethacrylate;polyvinylethylene; polyethyleneimine; poly(etherether)ketone;polyoxymethylene (POM); polyvinylphenol; polylactides;polymethacrylimide (PMI); polyalkenesulfone (PAS); polypropylethylene,polyethylene; polyhydroxyethylmethacrylate (HEMA); polydimethylsiloxane;polyacrylamide; polyimide; and block-copolymers. Preferred substratesfor the array include silicon, silica, glass, and polymers. Thesubstrate on which the patches reside may also be a combination of anyof the aforementioned substrate materials.

An array of the present invention may optionally further comprise acoating between the substrate and the organic thinfilm of its patches.This coating may either be formed on the substrate or applied to thesubstrate. The substrate can be modified with a coating by usingthin-film technology based, for instance, on physical vapor deposition(PVD), plasma-enhanced chemical vapor deposition (PECVD), or thermalprocessing. Alternatively, plasma exposure can be used to directlyactivate or alter the substrate and create a coating. For instance,plasma etch procedures can be used to oxidize a polymeric surface (forexample, polystyrene or polyethylene to expose polar functionalitiessuch as hydroxyls, carboxylic acids, aldehydes and the like) which thenacts as a coating.

The coating is optionally a metal film. Possible metal films includealuminum, chromium, titanium, tantalum, nickel, stainless steel, zinc,lead, iron, copper, magnesium, manganese, cadmium, tungsten, cobalt, andalloys or oxides thereof. In a preferred embodiment, the metal film is anoble metal film. Noble metals that may be used for a coating include,but are not limited to, gold, platinum, silver, and copper. In anespecially preferred embodiment, the coating comprises gold or a goldalloy. Electron-beam evaporation may be used to provide a thin coatingof gold on the surface of the substrate. In a preferred embodiment, themetal film is from about 50 nm to about 500 nm in thickness. In analternative embodiment, the metal film is from about 1 nm to about 1 μmin thickness.

In alternative embodiments, the coating comprises a composition selectedfrom the group consisting of silicon, silicon oxide, titania, tantalumoxide, silicon nitride, silicon hydride, indium tin oxide, magnesiumoxide, alumina, glass, hydroxylated surfaces, and polymers.

In one embodiment of the invention array, the surface of the coating isatomically flat. In this embodiment, the mean roughness of the surfaceof the coating is less than about 5 angstroms for areas of at least 25μm². In a preferred embodiment, the mean roughness of the surface of thecoating is less than about 3 angstroms for areas of at least 25 μm². Theultraflat coating can optionally be a template-stripped surface asdescribed in Hegner et al., Surface Science, 1993, 291:39-46 and Wagneret al., Langmuir, 1995, 11:3867-3875, both of which are incorporatedherein by reference.

It is contemplated that the coatings of many arrays will require theaddition of at least one adhesion layer between said coating and thesubstrate. Typically, the adhesion layer will be at least 6 angstromsthick and may be much thicker. For instance, a layer of titanium orchromium may be desirable between a silicon wafer and a gold coating. Inan alternative embodiment, an epoxy glue such as Epo-tek 377®, Epo-tek301-2®, (Epoxy Technology Inc., Billerica, Mass.) may be preferred toaid adherence of the coating to the substrate. Determinations as to whatmaterial should be used for the adhesion layer would be obvious to oneskilled in the art once materials are chosen for both the substrate andcoating. In other embodiments, additional adhesion mediators orinterlayers may be necessary to improve the optical properties of thearray, for instance, waveguides for detection purposes.

Deposition or formation of the coating (if present) on the substrate isperformed prior to the formation of the organic thinfilm thereon.Several different types of coating may be combined on the surface. Thecoating may cover the whole surface of the substrate or only parts ofit. The pattern of the coating may or may not be identical to thepattern of organic thinfilms used to immobilize the protein-captureagents. In one embodiment of the invention, the coating covers thesubstrate surface only at the site of the patches of protein-captureagents. Techniques useful for the formation of coated patches on thesurface of the substrate which are organic thinfilm compatible are wellknown to those of ordinary skill in the art. For instance, the patchesof coatings on the substrate may optionally be fabricated byphotolithography, micromolding (PCT Publication WO 96/29629), wetchemical or dry etching, or any combination of these.

The organic thinfilm on which each of the patches of protein-captureagents resides forms a layer either on the substrate itself or on acoating covering the substrate. The organic thinfilm on which theprotein-capture agents of the patches are immobilized is preferably lessthan about 20 nm thick. In some embodiments of the invention, theorganic thinfilm of each of the patches may be less than about 10 nmthick.

A variety of different organic thinfilms are suitable for use in thepresent invention. Methods for the formation of organic thinfilmsinclude in situ growth from the surface, deposition by physisorption,spin-coating, chemisorption, self-assembly, or plasma-initiatedpolymerization from gas phase. For instance, a hydrogel composed of amaterial such as dextran can serve as a suitable organic thinfilm on thepatches of the array. In one preferred embodiment of the invention, theorganic thinfilm is a lipid bilayer. In another preferred embodiment,the organic thinfilm of each of the patches of the array is a monolayer.A monolayer of polyarginine or polylysine adsorbed on a negativelycharged substrate or coating is one option for the organic thinfilm.Another option is a disordered monolayer of tethered polymer chains. Ina particularly preferred embodiment, the organic thinfilm is aself-assembled monolayer. The organic thinfilm is most preferably aself-assembled monolayer which comprises molecules of the formula X—R—Y,wherein R is a spacer, X is a functional group that binds R to thesurface, and Y is a functional group for binding protein-capture agentsonto the monolayer. In an alternative preferred embodiment, theself-assembled monolayer is comprised of molecules of the formula(X)_(a)R(Y)_(b) where a and b are, independently, integers greater thanor equal to 1 and X, R, and Y are as previously defined. In analternative preferred embodiment, the organic thinfilm comprises acombination of organic thinfilms such as a combination of a lipidbilayer immobilized on top of a self-assembled monolayer of molecules ofthe formula X—R—Y. As another example, a monolayer of polylysine canalso optionally be combined with a self-assembled monolayer of moleculesof the formula X—R—Y (see U.S. Pat. No. 5,629,213).

In all cases, the coating, or the substrate itself if no coating ispresent, must be compatible with the chemical or physical adsorption ofthe organic thinfilm on its surface. For instance, if the patchescomprise a coating between the substrate and a monolayer of molecules ofthe formula X—R—Y, then it is understood that the coating must becomposed of a material for which a suitable functional group X isavailable (see below). If no such coating is present, then it isunderstood that the substrate must be composed of a material for which asuitable functional group X is available.

In a preferred embodiment of the invention, the regions of the substratesurface, or coating surface, which separate the patches ofprotein-capture agents are free of organic thinfilm. In an alternativeembodiment, the organic thinfilm extends beyond the area of thesubstrate surface, or coating surface if present, covered by the patchesof protein-capture agents. For instance, optionally, the entire surfaceof the array may be covered by an organic thinfilm on which theplurality of spatially distinct patches of protein-capture agentsreside. An organic thinfilm which covers the entire surface of the arraymay be homogenous or may optionally comprise patches of differingexposed functionalities useful in the immobilization of patches ofdifferent protein-capture agents. In still another alternativeembodiment, the regions of the substrate surface or coating surface, ifa coating is present, between the patches of protein-capture agents arecovered by an organic thinfilm, but an organic thinfilm of a differenttype than that of the patches of protein-capture agents. For instance,the surfaces between the patches of protein-capture agents may be coatedwith an organic thinfilm characterized by low non-specific bindingproperties for proteins and other analytes.

A variety of techniques may be used to generate patches of organicthinfi m on the surface of the substrate or on the surface of a coatingon the substrate. These techniques are well known to those skilled inthe art and will vary depending upon the nature of the organic thinfilm,the substrate, and the coating if present. The techniques will also varydepending on the structure of the underlying substrate and the patternof any coating present on the substrate. For instance, patches of acoating which is highly reactive with an organic thinfilm may havealready been produced on the substrate surface. Arrays of patches oforganic thinfilm can optionally be created by microfluidics printing,microstamping (U.S. Pat. Nos. 5,512,131 and 5,731,152), or microcontactprinting (μCP) (PCT Publication WO 96/29629). Subsequent immobilizationof protein-capture agents to the reactive monolayer patches results intwo-dimensional arrays of the agents. Inkjet printer heads provideanother option for patterning monolayer X—R—Y molecules, or componentsthereof, or other organic thinfilm components to nanometer or micrometerscale sites on the surface of the substrate or coating (Lemmo et al.,Anal Chem., 1997, 69:543-551; U.S. Pat. Nos. 5,843,767 and 5,837,860).In some cases, commercially available arrayers based on capillarydispensing (for instance, OmniGrid™ from Genemachines, inc, San Carlos,Calif., and High-Throughput Microarrayer from IntelligentBio-Instruments, Cambridge, Mass.) may also be of use in directingcomponents of organic thinfilms to spatially distinct regions of thearray.

Diffusion boundaries between the patches of protein-capture agentsimmobilized on organic thinfilms such as self-assembled monolayers maybe integrated as topographic patterns (physical barriers) or surfacefunctionalities with orthogonal wetting behavior (chemical barriers).For instance, walls of substrate material or photoresist may be used toseparate some of the patches from some of the others or all of thepatches from each other. Alternatively, non-bioreactive organicthinfilms, such as monolayers, with different wettability may be used toseparate patches from one another.

In a preferred embodiment of the invention, each of the patches ofprotein-capture agents comprises a self-assembled monolayer of moleculesof the formula X—R—Y, as previously defined, and the patches areseparated from each other by surfaces free of the monolayer.

FIG. 1 shows the top view of one example of an array of patches reactivewith protein-capture agents. On the array, a number of patches 15 coverthe surface of the substrate 3.

FIG. 2 shows a detailed cross section of a patch 15 of the array of FIG.1. This view illustrates the use of a coating 5 on the substrate 3. Anadhesion interlayer 6 is also included in the patch. On top of the patchresides a self-assembled monolayer 7.

FIG. 3 shows a cross section of one row of the patches 15 of the arrayof FIG. 1. This figure also shows the use of a cover 2 over the array.Use of the cover 2 creates an inlet port 16 and an outlet port 17 forsolutions to be passed over the array.

A variety of chemical moieties may function as monolayer molecules ofthe formula X—R—Y in the array of the present invention. However, threemajor classes of monolayer formation are preferably used to expose highdensities of reactive omega-functionalities on the patches of the array:(i) alkylsiloxane monolayers (“silanes”) on hydroxylated andnon-hydroxylated surfaces (as taught in, for example, U.S. Pat. No.5,405,766, PCT Publication WO 96/38726, U.S. Pat. Nos. 5,412,087, and5,688,642); (ii) alkyl-thiol/dialkyldisulfide monolayers on noble metals(preferably Au(111)) (as, for example, described in Allara et al., U.S.Pat. No. 4,690,715; Bamdad et al., U.S. Pat. No. 5,620,850; Wagner etal., Biophysical Journal, 1996, 70:2052-2066); and (iii) alkyl monolayerformation on oxide-free passivated silicon (as taught in, for example,Linford et al., J. Am. Chem. Soc., 1995, 117:3145-3155, Wagner et al.,Journal of Structural Biology, 1997, 119:189-201, U.S. Pat. No.5,429,708). One of ordinary skill in the art, however, will recognizethat many possible moieties may be substituted for X, R, and/or Y,dependent primarily upon the choice of substrate, coating, and affinitytag. Many examples of monolayers are described in Ulman, An Introductionto Ultrathin Organic Films: From Langmuir-Blodgett to Self Assembly,Academic press (1991).

In one embodiment, the monolayer comprises molecules of the formula(X)_(a)R(Y)_(b) wherein a and b are, independently, equal to an integerbetween 1 and about 200. In a preferred embodiment, a and b are,independently, equal to an integer between 1 and about 80. In a morepreferred embodiment, a and b are, independently, equal to 1 or 2. In amost preferred embodiment, a and b are both equal to 1 (molecules of theformula X—R—Y).

If the patches of the invention array comprise a self-assembledmonolayer of molecules of the formula (X)_(a)R(Y)_(b), then R mayoptionally comprise a linear or branched hydrocarbon chain from about 1to about 400 carbons long. The hydrocarbon chain may comprise an alkyl,aryl, alkenyl, alkynyl, cycloalkyl, alkaryl, aralkyl group, or anycombination thereof. If a and b are both equal to one, then R istypically an alkyl chain from about 3 to about 30 carbons long. In apreferred embodiment, if a and b are both equal to one, then R is analkyl chain from about 8 to about 22 carbons long and is, optionally, astraight alkane. However, it is also contemplated that in an alternativeembodiment, R may readily comprise a linear or branched hydrocarbonchain from about 2 to about 400 carbons long and be interrupted by atleast one hetero atom. The interrupting hetero groups can include —O—,—CONH—, —CONHCO—, —NH—, —CSNH—, —CO—, —CS—, —S—, —SO—, —(OCH₂CH₂)_(n)—,(where n=1-20), —(CF₂)_(n)—, (where n=1-22), and the like.Alternatively, one or more of the hydrogen moieties of R can besubstituted with deuterium. In alternative, less preferred, embodiments,R may be more than about 400 carbons long.

X may be chosen as any group which affords chemisorption orphysisorption of the monolayer onto the surface of the substrate (or thecoating, if present). When the substrate or coating is a metal or metalalloy, X, at least prior to incorporation into the monolayer, can in oneembodiment be chosen to be an asymmetrical or symmetrical disulfide,sulfide, diselenide, selenide, thiol, isonitrile, selenol, a trivalentphosphorus compound, isothiocyanate, isocyanate, xanthanate,thiocarbamate, a phosphine, an amine, thio acid or a dithio acid. Thisembodiment is especially preferred when a coating or substrate is usedthat is a noble metal such as gold, silver, or platinum.

If the substrate of the array is a material such as silicon, siliconoxide, indium tin oxide, magnesium oxide, alumina, quartz, glass, orsilica, then the array of one embodiment of the invention comprises an Xthat, prior to incorporation into said monolayer, is a monohalosilane,dihalosilane, trihalosilane, trialkoxysilane, dialkoxysilane, or amonoalkoxysilane. Among these silanes, trichlorosilane andtrialkoxysilane are particularly preferred.

In a preferred embodiment of the invention, the substrate is selectedfrom the group consisting of silicon, silicon dioxide, indium tin oxide,alumina, glass, and titania; and X, prior to incorporation into saidmonolayer, is selected from the group consisting of a monohalosilane,dihalosilane, trihalosilane, trichlorosilane, trialkoxysilane,dialkoxysilane, monoalkoxysilane, carboxylic acids, and phosphates.

In another preferred embodiment of the invention, the substrate of thearray is silicon and X is an olefin.

In still another preferred embodiment of the invention, the coating (orthe substrate if no coating is present) is titania or tantalum oxide andX is a phosphate.

In other embodiments, the surface of the substrate (or coating thereon)is composed of a material such as titanium oxide, tantalum oxide, indiumtin oxide, magnesium oxide, or alumina where X is a carboxylic acid oralkylphosphoric acid. Alternatively, if the surface of the substrate (orcoating thereon) of the array is copper, then X may optionally be ahydroxamic acid.

If the substrate used in the invention is a polymer, then in many casesa coating on the substrate such as a copper coating will be included inthe array. An appropriate functional group X for the coating would thenbe chosen for use in the array. In an alternative embodiment comprisinga polymer substrate, the surface of the polymer may be plasma-modifiedto expose desirable surface functionalities for monolayer formation. Forinstance, EP 780423 describes the use of a monolayer molecule that hasan alkene X functionality on a plasma exposed surface. Still anotherpossibility for the invention array comprised of a polymer is that thesurface of the polymer on which the monolayer is formed isfunctionalized by copolymerization of appropriately functionalizedprecursor molecules.

Another possibility is that prior to incorporation into the monolayer, Xcan be a free-radical-producing moiety. This functional group isespecially appropriate when the surface on which the monolayer is formedis a hydrogenated silicon surface. Possible free-radical producingmoieties include, but are not limited to, diacylperoxides, peroxides,and azo compounds. Alternatively, unsaturated moieties such asunsubstituted alkenes, alkynes, cyano compounds and isonitrile compoundscan be used for X, if the reaction with X is accompanied by ultraviolet,infrared, visible, or microwave radiation.

In alternative embodiments, X, prior to incorporation into themonolayer, may be a hydroxyl, carboxyl, vinyl, sulfonyl, phosphoryl,silicon hydride, or an amino group.

The component, Y, of the monolayer is a functional group responsible forbinding a protein-capture agent onto the monolayer. In a preferredembodiment of the invention, the Y group is either highly reactive(activated) towards the protein-capture agent (or its affinity tag) oris easily converted into such an activated form. In a preferredembodiment, the coupling of Y with the protein-capture agent occursreadily under normal physiological conditions not detrimental to theability of the protein-capture agent to bind its binding partner. Thefunctional group Y may either form a covalent linkage or a noncovalentlinkage with the protein-capture agent (or its affinity tag, ifpresent). In a preferred embodiment, the functional group Y forms acovalent linkage with the protein-capture agent or its affinity tag. Itis understood that following the attachment of the protein-capture agent(with or without an affinity tag) to Y, the chemical nature of Y mayhave changed. Upon attachment of the protein-capture agent, Y may evenhave been removed from the organic thinfilm.

In one embodiment of the array of the present invention, Y is afunctional group that is activated in situ. Possibilities for this typeof functional group include, but are not limited to, such simplemoieties such as a hydroxyl, carboxyl, amino, aldehyde, carbonyl,methyl, methylene, alkene, alkyne, carbonate, aryliodide, or a vinylgroup. Appropriate modes of activation would be obvious to one skilledin the art. Alternatively, Y can comprise a functional group thatrequires photoactivation prior to becoming activated enough to trap theprotein-capture agent.

In an especially preferred embodiment of the array of the presentinvention, Y is a complex and highly reactive functional moiety that iscompatible with monolayer formation and needs no in situ activationprior to reaction with the protein-capture agent and/or affinity tag.Such possibilities for Y include, but are not limited to, maleimide,N-hydroxysuccinimide (Wagner et al., Biophysical Journal, 1996,70:2052-2066), nitrilotriacetic acid (U.S. Pat, No. 5,620,850),activated hydroxyl, haloacetyl, bromoacetyl, iodoacetyl, activatedcarboxyl, hydrazide, epoxy, aziridine, sulfonylchloride,trifluoromethyldiaziridine, pyridyldisulfide, N-acyl-imidazole,imidazolecarbamate, vinylsulfone, succinimidylcarbonate, arylazide,anhydride, diazoacetate, benzophenone, isothiocyanate, isocyanate,imidoester, fluorobenzene, and biotin.

FIG. 4 shows one example of a monolayer on a substrate 3. In thisexample, substrate 3 comprises glass. The monolayer is thiolreactivebecause it bears a maleimidyl functional group Y.

FIG. 5 shows another example of a monolayer on a substrate 3 which issilicon. In this case, however, a thinfilm gold coating 5 covers thesurface of the substrate 3. Also, in this embodiment, a titaniumadhesion interlayer 6 is used to adhere the coating 5 to the substrate3. This monolayer is aminoreactive because it bears anN-hydroxysuccinimidyl functional group Y.

In an alternative embodiment, the functional group Y of the array isselected from the group of simple functional moieties. Possible Yfunctional groups include, but are not limited to, —OH, —NH₂, —COOH,—COOR, —RSR, —PO₄ ⁻³, —OSO₃ ⁻², —SO₃ ⁻, —COO⁻, —SOO⁻, —CONR₂, —CN, —NR₂,and the like.

The monolayer molecules of the present invention can optionally beassembled on the surface in parts. In other words, the monolayer neednot necessarily be constructed by chemisorption or physisorption ofmolecules of the formula X—R—Y to the surface of the substrate (orcoating). Instead, in one embodiment, X may be chemisorbed orphysisorbed to the surface of the substrate (or coating) alone first.Then, R or even just individual components of R can be attached to Xthrough a suitable chemical reaction. Upon completion of addition of thespacer R to the X moiety already immobilized on the surface, Y can beattached to the ends of the monolayer molecule through a suitablecovalent linkage.

Not all self-assembled monolayer molecules on a given patch need beidentical to one another. Some patches may comprise mixed monolayers.For instance, the monolayer of an individual patch may optionallycomprise at least two different molecules of the formula X—R—Y, aspreviously described. This second X—R—Y molecule may immobilize the sameor a different protein-capture agent having the same binding partner asthe first. In addition, some of the monolayer molecules X—R—Y of a patchmay have failed to attach any protein-capture agent.

As another alternative embodiment of the invention, a mixed,self-assembled monolayer of an individual patch on the array maycomprise both molecules of the formula X—R—Y, as previously described,and molecules of the formula, X—R—V where R is a spacer, X is afunctional group that binds R to the surface, and V is a moiety which isbiocompatible with proteins and resistant to the non-specific binding ofproteins. For example, V may consist of a hydroxyl, saccharide, oroligo/polyethylene glycol moiety (EP Publication 780423).

In still another embodiment of the invention, the array comprises atleast one unreactive patch of organic thinfilm on the substrate orcoating surface which is devoid of any protein-capture agent. Forinstance, the unreactive patch may optionally comprise a monolayer ofmolecules of the formula X—R—V, where R is a spacer, X is a functionalgroup that binds R to the surface, and V is a moiety resistant to thenon-specific binding of proteins. The unreactive patch may serve as acontrol patch or be useful in background binding measurements.

Regardless of the nature of the monolayer molecules, in some arrays itmay be desirable to provide crosslinking between molecules of anindividual patch's monolayer. In general, crosslinking confersadditional stability to the monolayer. Such methods are familiar tothose skilled in the art (for instance, see Ulman, An Introduction toUltrathin Organic Films: From Langmuir-Blodgett to Self-Assembly,Academic Press (1991)).

After completion of formation of the monolayer on the patches, theprotein-capture agent may be attached to the monolayer via interactionwith the Y-functional group. Y-functional groups which fail to reactwith any protein-capture agents are preferably quenched prior to use ofthe array. Quenching may be performed using small molecular weightagents such as glycine with NHS coupling schemes, however a particularlypreferred embodiment includes quenching un-reacted Y functional groupswith polymeric molecules having non-specific protein binding resistantproperties such as polyethylene glycol or PEG. For example, a surfacedisplaying NHS or N-hydroxysuccinamide groups is first reacted with aprotein capture agent such as biotin or an antibody or antibody-likemolecule, then, the un-reacted NHS Y functional groups are then reactedto amine-PEG to quench the NHS group. An example of a final chip productproduced by this method would include a surface, an ordered hydrophobicmonolayer formed from hydrocarbon polymer chains attached to thesurface, a hydrophilic monolayer formed from hydrophilic polymer chainsattached to the hydrophobic monolayer, protein capture agents attachedto one or more of the hydrophilic polymer chains of the hydrophilicmonolayer, and PEG attached to one or more of any hydrophilic polymerchains lacking a protein capture agent.

FIG. 10A depicts chip (1001) with thinfilm (1003) formed fromhydrophobic monolayer (1005) attached to surface (1005) of chip (1001)and hydrophilic monolayer (1007) attached to hydrophobic monolayer(1005). Functional groups (Y) are attached to the end of hydrophilicchains (1011) away from chip surface (1005) to form a functional groupbearing surface (1017) outwardly facing away from chip surface (1005).

FIG. 10B depicts how a first subset (Y1) of functional groups (Y) arereacted with protein capture agents (PCA) to immobilize such agents tochip (1001).

FIG. 10C depicts how a second subset (Y2) of functional groups (Y),which is mutually exclusive of first subset (Y1), is reacted with aquenching agent (Q) to quench unreacted functional groups of secondsubset (1023).

(d) Affinity Tags and Immobilization of Protein-capture Agents

In a preferred embodiment, the protein-immobilizing patches of the arrayfurther comprise an affinity tag that enhances immobilization of theprotein-capture agent onto the organic thinfilm. The use of an affinitytag on the protein-capture agent of the array typically provides severaladvantages. An affinity tag can confer enhanced binding or reaction ofthe protein-capture agent with the functionalities on the organicthinfilm, such as Y if the organic thinfilm is a an X—R—Y monolayer aspreviously described. This enhancement effect may be either kinetic orthermodynamic. The affinity tag/thinfilm combination used in the patchesof the array preferably allows for immobilization of the protein-captureagents in a manner which does not require harsh reaction conditions thatare adverse to protein stability or function. In most embodiments,immobilization to the organic thinfilm in aqueous, biological buffers isideal.

An affinity tag also preferably offers immobilization on the organicthinfilm that is specific to a designated site or location on theprotein-capture agent (site-specific immobilization). For this to occur,attachment of the affinity tag to the protein-capture agent must besite-specific. Site-specific immobilization helps ensure that theprotein-binding site of the agent, such as the antigen-binding site ofthe antibody moiety, remains accessible to ligands in solution. Anotheradvantage of immobilization through affinity tags is that it allows fora common immobilization strategy to be used with multiple, differentprotein-capture agents.

The affinity tag is optionally attached directly, either covalently ornoncovalently, to the protein-capture agent. In an alternativeembodiment, however, the affinity tag is either covalently ornoncovalently attached to an adaptor which is either covalently ornoncovalently attached to the protein-capture agent.

In a preferred embodiment, the affinity tag comprises at least one aminoacid. The affinity tag may be a polypeptide comprising at least twoamino acids which is reactive with the functionalities of the organicthinfilm. Alternatively, the affinity tag may be a single amino acidwhich is reactive with the organic thinfilm. Examples of possible aminoacids which could be reactive with an organic thinfilm include cysteine,lysine, histidine, arginine, tyrosine, aspartic acid, glutamic acid,tryptophan, serine, threonine, and glutamine. A polypeptide or aminoacid affinity tag is preferably expressed as a fusion protein with theprotein-capture agent when the protein-capture agent is a protein, suchas an antibody or antibody fragment. Amino acid affinity tags provideeither a single amino acid or a series of amino acids that can interactwith the functionality of the organic thinfilm, such as the Y-functionalgroup of the self-assembled monolayer molecules. Amino acid affinitytags can be readily introduced into recombinant proteins to facilitateoriented immobilization by covalent binding to the Y-functional group ofa monolayer or to a functional group on an alternative organic thinfilm.

The affinity tag may optionally comprise a poly(amino acid) tag. Apoly(amino acid) tag is a polypeptide that comprises from about 2 toabout 100 residues of a single amino acid, optionally interrupted byresidues of other amino acids. For instance, the affinity tag maycomprise a poly-cysteine, polylysine, poly-arginine, or poly-histidine.Amino acid tags are preferably composed of two to twenty residues of asingle amino acid, such as, for example, histidines, lysines, arginines,cysteines, glutamines, tyrosines, or any combination of these. Accordingto a preferred embodiment, an amino acid tag of one to twenty aminoacids includes at least one to ten cysteines for thioether linkage; orone to ten lysines for amide linkage; or one to ten arginines forcoupling to vicinal dicarbonyl groups. One of ordinary skill in the artcan readily pair suitable affinity tags with a given functionality on anorganic thinfilm.

The position of the amino acid tag can be at an amino-, orcarboxy-terminus of the protein-capture agent which is a protein, oranywhere in-between, as long as the protein-binding region of theprotein-capture agent, such as the antigen-binding region of animmobilized antibody moiety, remains in a position accessible forprotein binding. Where compatible with the protein-capture agent chosen,affinity tags introduced for protein purification are preferentiallylocated at the C-terminus of the recombinant protein to ensure that onlyfull-length proteins are isolated during protein purification. Forinstance, if intact antibodies are used on the arrays, then theattachment point of the affinity tag on the antibody is preferablylocated at a C-terminus of the effector (Fc) region of the antibody. IfscFvs are used on the arrays, then the attachment point of the affinitytag is also preferably located at the C-terminus of the molecules.

Affinity tags may also contain one or more unnatural amino acids.Unnatural amino acids can be introduced using suppressor tRNAs thatrecognize stop codons (i.e., amber) (Noren et al., Science, 1989,244:182-188; Ellman et al., Methods Enzym., 1991, 202:301-336; Cload etal., Chem. Biol., 1996, 3:1033-1038). The tRNAs are chemicallyamino-acylated to contain chemically altered (“unnatural”) amino acidsfor use with specific coupling chemistries (i.e., ketone modifications,photoreactive groups).

In an alternative embodiment the affinity tag can comprise an intactprotein, such as, but not limited to, glutathione S-transferase, anantibody, avidin, or streptavidin.

When the protein-capture agent is a protein and the affinity tag is aprotein, such as a poly(amino acid) tag, or a single amino acid, theaffinity tag is preferably attached to the protein-capture agent bygenerating a fusion protein. Alternatively, protein synthesis or proteinligation techniques known to those skilled in the art may be used. Forinstance, intein-mediated protein ligation may optionally be used toattach the affinity tag to the protein-capture agent (Mathys, et al.,Gene 231:1-13, 1999; Evans, et al., Protein Science 7:2256-2264, 1998).

Other protein conjugation and immobilization techniques known in the artmay be adapted for the purpose of attaching affinity tags to theprotein-capture agent. For instance, in an alternative embodiment of thearray, the affinity tag may be an organic bioconjugate which ischemically coupled to the protein-capture agent of interest. Biotin orantigens may be chemically cross linked to the protein. Alternatively, achemical crosslinker may be used that attaches a simple functionalmoiety such as a thiol or an amine to the surface of a protein servingas a protein-capture agent on the array.

In an alternative embodiment of the invention, the organic thinfilm ofeach of the patches comprises, at least in part, a lipid monolayer orbilayer, and the affinity tag comprises a membrane anchor.

FIG. 6 shows a detailed cross section of a patch on one embodiment ofthe invention array. In this embodiment, a protein-capture agent 10 isimmobilized on a monolayer 7 on a substrate 3. An affinity tag 8connects the protein-capture agent 10 to the monolayer 7. The monolayer7 is formed on a coating 5 which is separated from the substrate 3 by aninterlayer 6.

In an alternative embodiment of the invention, no affinity tag is usedto immobilize the protein-capture agents onto the organic thinfilm. Anamino acid or other moiety (such as a carbohydrate moiety) inherent tothe protein-capture agent itself may instead be used to tether theprotein-capture agent to the reactive group of the organic thinfilm. Inpreferred embodiments, the immobilization is site-specific with respectto the location of the site of immobilization on the protein-captureagent. For instance, the sulfhydryl group on the C-terminal region ofthe heavy chain portion of a Fab′ fragment generated by pepsin digestionof an antibody, followed by selective reduction of the disulfide betweenmonovalent Fab′ fragments, may be used as the affinity tag.Alternatively, a carbohydrate moiety on the Fc portion of an intactantibody can be oxidized under mild conditions to an aldehyde groupsuitable for immobilizing the antibody on a monolayer via reaction witha hydrazide-activated Y group on the monolayer. Examples ofimmobilization of protein-capture agents without any affinity tag in asite-specific manner can be found in Dammer et al., Biophys J.,70:2437-2441, 1996 and the specific examples, Examples 5-7, below.

Since the protein-capture agents of at least some of the differentpatches on the array are different from each other, different solutions,each containing a different, preferably, affinity-tagged protein-captureagent, must be delivered to their individual patches. Solutions ofprotein-capture agents may be transferred to the appropriate patches viaarrayers which are well-known in the art and even commerciallyavailable. For instance, microcapillary-based dispensing systems may beused. These dispensing systems are preferably automated andcomputer-aided. A description of and building instructions for anexample of a microarrayer comprising an automated capillary system canbe found on the internet at http://cmgm.stanford.edu/pbrown/array.htmland http://cmgm.stanford.edu/pbrown/mguide/index.html. The use of othermicroprinting techniques for transferring solutions containing theprotein-capture agents to the agent-reactive patches is also possible.Ink-jet printer heads may also optionally be used for precise deliveryof the protein-capture agents to the agent-reactive patches.Representative, non-limiting disclosures of techniques useful fordepositing the protein-capture agents on the patches may be found, forexample, in U.S. Pat. Nos. 5,731,152 (stamping apparatus), U.S. Pat. No.5,807,522 (capillary dispensing device), U.S. Pat. No. 5,837,860(ink-jet printing technique, Hamilton 2200 robotic pipetting deliverysystem), and U.S. Pat. No. 5,843,767 (ink-jet printing technique,Hamilton 2200 robotic pipetting delivery system), all incorporated byreference herein.

(e) Adaptors

Another embodiment of the array of the present invention comprises anadaptor that links the affinity tag to the protein-capture agent on thepatches of the array. The additional spacing of the protein-captureagent from the surface of the substrate (or coating) that is afforded bythe use of an adaptor is particularly advantageous if theprotein-capture agent is a protein, since proteins are known to be proneto surface inactivation. The adaptor may optionally afford someadditional advantages as well. For instance, the adaptor may helpfacilitate the attachment of the protein-capture agent to the affinitytag. In another embodiment, the adaptor may help facilitate the use of aparticular detection technique with the array. One of ordinary skill inthe art will be able to choose an adaptor which is appropriate for agiven affinity tag. For instance, if the affinity tag is streptavidin,then the adaptor could be biotin that is chemically conjugated to theprotein-capture agent which is to be immobilized.

In one embodiment, the adaptor comprises a protein. In anotherembodiment, the affinity tag, adaptor, and protein-capture agenttogether compose a fusion protein. Such a fusion protein may be readilyexpressed using standard recombinant DNA technology. Adaptors which areproteins are especially useful to increase the solubility of theprotein-capture agent of interest and to increase the distance betweenthe surface of the substrate or coating and the protein-capture agent.Use of a protein adaptor can also be very useful in facilitating thepreparative steps of protein purification by affinity binding prior toimmobilization on the array. Examples of possible adaptor proteinsinclude glutathione-S-transferase (GST), maltose-binding protein,chitin-binding protein, thioredoxin, green-fluorescent protein (GFP).GFP can also be used for quantification of surface binding. In apreferred embodiment, when the protein-capture agent is an antibodymoiety comprising the Fc region, the adaptor is a polypeptide, such asprotein G, protein A, or recombinant protein A/G (a gene fusion productsecreted from a non-pathogenic form of Bacillus which contains four Fcbinding domains from protein A and two from protein G).

FIG. 7 shows a cross section of a patch on one particular embodiment ofthe invention array. The patch comprises a protein-capture agent 10immobilized on a monolayer 7 via both an affinity tag 8 and an adaptor9. The monolayer 7 rests on a coating 5. An interlayer 6 is used betweenthe coating 5 and the substrate 3.

(f) Preparation of the Protein-Capture Agents of the Array

The protein-capture agents used on the array may be produced by any ofthe variety of means known to those of ordinary skill in the art. In apreferred embodiment of the invention, the protein-capture agents areproteins, and in an especially preferred embodiment, the protein-captureagents are antibodies or antibody fragments. Therefore, methods ofpreparing these types of possible protein-capture agents are emphasizedhere.

In preparation for immobilization to the arrays of the presentinvention, the antibody moiety, or any other protein-capture agent whichis a protein or polypeptide, can optionally be expressed fromrecombinant DNA either in vivo or in vitro. The cDNA of the antibody orantibody fragment or other protein-capture agent is cloned into anexpression vector (many examples of which are commercially available)and introduced into cells of the appropriate organism for expression. Abroad range of host cells and expression systems may be used to producethe antibodies and antibody fragments, or other proteins, which serve asthe protein-capture agents on the array. Expression in vivo may be donein bacteria (for example, Escherichia coli), plants (for example,Nicotiana tabacum), lower eukaryotes (for example, Saccharomycescerevisiae, Saccharomyces pombe, Pichia pastoris), or higher eukaryotes(for example, bacculovirus-infected insect cells, insect cells,mammalian cells). For in vitro expression PCR-amplified DNA sequencesare directly used in coupled in vitro transcription/translation systems(for instance: Escherichia coli S30 lysates from T7 RNA polymeraseexpressing, preferably protease-deficient strains; wheat germ lysates;reticulocyte lysates (Promega, Pharmacia, Panvera)). The choice oforganism for optimal expression depends on the extent ofpost-translational modifications (i.e., glycosylation,lipid-modifications) desired. The choice of expression system alsodepends on other issues, such as whether an intact antibody is to beproduced or just a fragment of an antibody (and which fragment), sincedisulfide bond formation will be affected by the choice of a host cell.One of ordinary skill in the art will be able to readily choose whichhost cell type is most suitable for the protein-capture agent andapplication desired.

DNA sequences encoding affinity tags and adaptors can be engineered intothe expression vectors such that the protein-capture agent genes ofinterest can be cloned in frame either 5′ or 3′ of the DNA sequenceencoding the affinity tag and adaptor protein.

The expressed protein-capture agents are purified by affinitychromatography using commercially available resins.

Preferably, production of a plurality of protein-capture agents involvesparallel processing from cloning to protein expression and proteinpurification. cDNAs for the protein-capture agent of interest will beamplified by PCR using cDNA libraries or expressed sequence tags (EST)clones as templates. For in vivo expression of the proteins, cDNAs canbe cloned into commercial expression vectors (Qiagen, Novagen, Clontech)and introduced into an appropriate organism for expression (see above).For in vitro expression PCR-amplified DNA sequences are directly used incoupled in vitro transcription/translation systems (see above).

Escherichia coli-based protein expression is generally the method ofchoice for soluble proteins that do not require extensivepost-translational modifications for activity. Extracellular orintracellular domains of membrane proteins will be fused to proteinadaptors for expression and purification.

The entire approach can be performed using 96-well assay plates. PCRreactions are carried out under standard conditions. Oligonucleotideprimers contain unique restriction sites for facile cloning into theexpression vectors. Alternatively, the TA cloning system (Clontech) canbe used. The expression vectors contain the sequences for affinity tagsand the protein adaptors. PCR products are ligated into the expressionvectors (under inducible promoters) and introduced into the appropriatecompetent Escherichia coli strain by calcium-dependent transformation(strains include: XL-1 blue, BL21, SG13009(lon-)). TransformedEscherichia coli cells are plated and individual colonies transferredinto 96-array blocks. Cultures are grown to mid-log phase, induced forexpression, and cells collected by centrifugation. Cells are resuspendedcontaining lysozyme and the membranes broken by rapid freeze/thawcycles, or by sonication. Cell debris is removed by centrifugation andthe supernatants transferred to 96-tube arrays. The appropriate affinitymatrix is added, the protein-capture agent of interest is bound andnonspecifically bound proteins are removed by repeated washing stepsusing 12-96 pin suction devices and centrifugation. Alternatively,magnetic affinity beads and filtration devices can be used (Qiagen). Theproteins are eluted and transferred to a new 96-well array. Proteinconcentrations are determined and an aliquot of each protein-captureagent is spotted onto a nitrocellulose filter and verified by Westernanalysis using an antibody directed against the affinity tag on theprotein-capture agent. The purity of each sample is assessed by SDS-PAGEand Silver staining or mass spectrometry. The protein-capture agents arethen snap-frozen and stored at −80° C.

Saccharomyces cerevisiae allows for the production of glycosylatedprotein-capture agents such as antibodies or antibody fragments. Forproduction in Saccharomyces cerevisiae, the approach described above forEscherichia coli can be used with slight modifications fortransformation and cell lysis. Transformation of Saccharomycescerevisiae is by lithium-acetate and cell lysis is either by lyticasedigestion of the cell walls followed by freeze-thaw, sonication orglass-bead extraction. Variations of post-translational modificationscan be obtained by using different yeast strains (i.e., Saccharomycespombe, Pichia pastoris).

One aspect of the bacculovirus system is the array of post-translationalmodifications that can be obtained, although antibodies and otherproteins produced in bacculovirus contain carbohydrate structures verydifferent from those produced by mammalian cells. Thebacculovirus-infected insect cell system requires cloning of viruses,obtaining high titer stocks and infection of liquid insect cellsuspensions (cells such as SF9, SF21).

Mammalian cell-based expression requires transfection and cloning ofcell lines. Either lymphoid or non-lymphoid cell may be used in thepreparation of antibodies and antibody fragments. Soluble proteins suchas antibodies are collected from the medium while intracellular ormembrane bound proteins require cell lysis (either detergentsolubilization, freeze-thaw). The protein-capture agents can then bepurified analogous to the procedure described for Escherichia coli.

For in vitro translation the system of choice is Escherichia colilysates obtained from protease-deficient and T7 RNA polymeraseoverexpressing strains. Escherichia coli lysates provide efficientprotein expression (30-50 μg/ml lysate). The entire process is carriedout in 96-well arrays. Antibody genes or other protein-capture agentgenes of interest are amplified by PCR using oligonucleotides thatcontain the gene-specific sequences containing a T7 RNA polymerasepromoter and binding site and a sequence encoding the affinity tag.Alternatively, an adaptor protein can be fused to the gene of interestby PCR. Amplified DNAs can be directly transcribed and translated in theEscherichia coli lysates without prior cloning for fast analysis. Theantibody fragments or other proteins are then isolated by binding to anaffinity matrix and processed as described above.

Alternative in vitro translation systems which may be used include wheatgerm extracts and reticulocyte extracts. In vitro synthesis of membraneproteins or post-translationally modified proteins will requirereticulocyte lysates in combination with microsomes.

In one embodiment of the invention, the protein-capture agents on thearray are monoclonal antibodies. The production of monoclonal antibodiesagainst specific protein targets is routine using standard hybridomatechnology. In fact, numerous monoclonal antibodies are availablecommercially. The preparation and use of an array of monoclonalantibodies is illustrated in the specific example, Example 8, below.

As an alternative to obtaining antibodies or antibody fragments by cellfusion or from continuous cell lines, the antibody moieties may beexpressed in bacteriophage. Such antibody phage display technologies arewell known to those skilled in the art. The bacteriophage expressionsystems allow for the random recombination of heavy- and light-chainsequences, thereby creating a library of antibody sequences which can beselected against the desired antigen. The expression system can be basedon bacteriophage λ or, more preferably, on filamentous phage. Thebacteriophage expression system can be used to express Fab fragments,Fv's with an engineered intermolecular disulfide bond to stabilize theV_(H)-V_(L) pair (dsFv's), scFvs, or diabody fragments.

The antibody genes of the phage display libraries may be frompre-immunized donors. For instance, the phage display library could be adisplay library prepared from the spleens of mice previously immunizedwith a mixture of proteins (such as a lysate of human T-cells).Immunization can optionally be used to bias the library to contain agreater number of recombinant antibodies reactive towards a specific setof proteins (such as proteins found in human T-cells). Alternatively,the library antibodies may be derived from naive or synthetic libraries.The naive libraries have been constructed from spleens of mice whichhave not been contacted by external antigen. In a synthetic library,portions of the antibody sequence, typically those regions correspondingto the complementarity determining regions (CDR) loops, have beenmutagenized or randomized.

The phage display method involves batch-cloning the antibody genelibrary into a phage genome as a fusion to the gene encoding one of thephage coat proteins (pIII, pVI, or pVIII). The pIII phage protein geneis preferred. When the fusion product is expressed it is incorporatedinto the mature phage coat. As a result, the antibody is displayed as afusion on the surface of the phage and is available for binding andhence, selection, on a target protein. Once a phage particle is selectedas bearing an antibody-coat protein fusion with the desired affinitytowards the target protein, the genetic material within the phageparticle which corresponds to the displayed antibody can be amplifiedand sequenced or otherwise analyzed.

In a preferred embodiment, a phagemid is used as the expression vectorin the phage display procedures. A phagemid is a small plasmid vectorthat carries gene III with appropriate cloning sites and a phagepackaging signal and contains both host and phage origins ofreplication. The phagemid is unable to produce a complete phage as thegene III fusion is the only phage gene encoded on the phagemid. A viablephage can be produced by infecting cells containing the phagemid with ahelper phage containing a defective replication origin. A hybrid phageemerges which contains all of the helper phage proteins as well as thegene III-rAb fusion. The emergent phage contains the phagemid DNA only.

In a preferred embodiment of the invention, the recombinant antibodiesused in phage display methods of preparing protein-capture agents forthe arrays of the invention are expressed as genetic fusions to thebacteriophage gene III protein on a phagemid vector. For instance, theantibody variable regions encoding a single-chain Fv fragment can befused to the amino terminus of the gene III protein on a phagemid.Alternatively, the antibody fragment sequence could be fused to theamino terminus of a truncated pIII sequence lacking the first twoN-terminal domains. The phagemid DNA encoding the antibody-pIII fusionis preferably packaged into phage particles using a helper phage such asM13KO7 or VCS-M13, which supplies all structural phage proteins.

To display Fab fragments on phage, either the light or heavy (Fd) chainis fused via its C-terminus to pIII. The partner chain is expressedwithout any fusion to pIII so that both chains can associate to form anintact Fab fragment.

Any method of selection may be used which separates those phageparticles which do bind the target protein from those which do not. Theselection method must also allow for the recovery of the selectedphages. Most typically, the phage particles are selected on animmobilized target protein. Some phage selection strategies known tothose skilled in the art include the following: panning on animmobilized antigen; panning on an immobilized antigen using specificelution; using biotinylated antigen and then selecting on a streptavidinresin or streptavidin-coated magnetic beads; affinity purification;selection on Western blots (especially useful for unknown antigens orantigens difficult to purify); in vivo selection; and pathfinderselection. If the selected phage particles are amplified betweenselection rounds, multiple iterative rounds of selection may optionallybe performed.

Elution techniques will vary depending upon the selection processchosen, but typical elution techniques include washing with one of thefollowing solutions: HCl or glycine buffers; basic solutions such astriethylamine; chaotropic agents; solutions of increased ionic strength;or DTT when biotin is linked to the antigen by a disulfide bridge. Othertypical methods of elution include enzymatically cleaving a proteasesite engineered between the antibody and gene III, or by competing forbinding with excess antigen or excess antibodies to the antigen.

A method for producing an array of antibody fragments thereforecomprises first selecting recombinant bacteriophage which expressantibody fragments from a phage display library. The recombinantbacteriophage are selected by affinity binding to a protein which is anexpression product, or fragment thereof, of a cell or population ofcells in an organism. (Iterative rounds of selection are possible, butoptional.) Next, at least one purified sample of an antibody fragmentfrom a bacteriophage which was selected in the first step is produced.This antibody production step typically entails infecting E. coli cellswith the selected bacteriophage. In the absence of helper phage, theselected bacteriophage then replicate as expressive plasmids withoutproducing phage progeny. Alternatively, the antibody fragment gene ofthe selected recombinant bacteriophage is isolated, amplified, and thenexpressed in a suitable expression system. In either case, followingamplification, the expressed antibody fragment of the selected andamplified recombinant bacteriophage is isolated and purified. In a thirdstep of the method, the earlier steps of phage display selection andpurified antibody fragment production are repeated using affinitybinding to different proteins which are expression products, orfragments thereof, of the same cell or population of cells as beforeuntil the desired plurality of purified samples of different antibodieswith different binding pairs are produced. In a final step of themethod, the antibody fragment of each different purified sample isimmobilized onto an organic thinfilm on a separate patch on the surfaceof a substrate to form a plurality of patches of antibody fragments ondiscrete, known regions of the substrate surface covered by organicthinfilm.

For instance, to generate an antibody array with antibody fragmentsagainst known protein targets, open reading frames of the known proteintargets identified in DNA databases are amplified by polymerase chainreaction and transcribed and translated in vitro to produce proteins onwhich a recombinant bacteriophage expressing single-chain antibodyfragments are selected. Once selected, the antibody fragment sequence ofthe selected bacteriophage is amplified (typically using the polymerasechain method) and recloned into a desirable expression system. Theexpressed antibody fragments are purified and then printed onto organicthinfilms on substrates to form the high density arrays.

In another embodiment of the invention, a method for producing an arrayof protein-capture agents is provided which comprises first selectingprotein-capture agents from a library of protein-capture agents, wherethe protein-capture agents are selected by their affinity binding to theproteins from a cellular extract or body fluid. Preferably, the proteinsare from a cellular extract. The proteins from the cellular extract orbody fluid would typically be immobilized prior to the selection step.Suitable methods of immobilization such as crosslinking of the proteinsto a resin are well known to one of ordinary skill in the art. The nextstep of this method comprises producing a plurality of purified samplesof the selected protein-capture agents. The protein-capture agent ofeach different purified sample is immobilized onto an organic thinfilmon a separate patch on the surface of a substrate to form a plurality ofpatches of protein-capture agents on discrete, known regions of thesubstrate surface covered by organic thinfilm.

This method of array preparation optionally also comprises theadditional step of biasing the library of protein-capture agents byeliminating from the library those protein-capture agents which bindcertain proteins, such as the proteins of a second cellular extract,wherein the protein-capture agents which are eliminated are removed fromthe library by their binding affinity to those certain proteins. Thisstep of biasing the library may optionally occur after the selectionstep by affinity binding to the protein, but more typically, it occursprior to that selection step. The order of the selecting and biasingsteps will depend on the nature of the selection and elution proceduresused in the method. One of ordinary skill in the art will readily beable to determine an appropriate series of steps.

In one embodiment of the optional step of biasing the library ofprotein-capture agents, the library is biased to eliminateprotein-capture agents that recognize common proteins or proteins ofnon-interest. This is typically achieved by passing the library over anaffinity surface, such as a chromatography column, containingcross-linked proteins of non-interest. The “flowthrough” containingprotein-capture agents that did not react with the affinity surface iscollected. This procedure enriches the library for protein-captureagents which bind proteins of interest or proteins specific to the cellto be assayed. For instance, if the library is derived from a specificcell type such a a human T-cell, the library may optionally be biased bypassing it over an affinity surface which contains proteins preparedfrom a lysate of human fibroblasts or bacterial proteins to enrich thelibrary for protein-capture agents which bind proteins specificallypresent in fibroblasts.

In a preferred embodiment of the method of preparing the array ofprotein-capture agents described above, the protein-capture agents areantibody fragments displayed on the surface of recombinantbacteriophages and the library of protein-capture agents is a phagedisplay library. Therefore, a method for producing an antibody arraycomprises first selecting recombinant bacteriophage expressing antibodyfragments from a phage display library, where the bacteriophage areselected by affinity binding to immobilized proteins of a body fluid, ormore preferably, a cellular extract. The next step of this methodcomprises producing a plurality of purified samples of antibodyfragments expressed by the selected recombinant bacteriophage.Preferably, antibody fragments which specifically bind more than 1000 ofthe proteins of the cellular extract are produced in this manner. In afinal step of the method, the antibody fragment of each differentpurified sample is immobilized onto an organic thinfilm on a separatepatch on the surface of a substrate to form a plurality of patches ofantibody fragments on discrete known regions of the substrate surface.One specific example of this method is outlined in Example 6, below.Again, this method optionally also comprises the additional step ofbiasing the phage display library by eliminating from the library thosebacteriophage displaying antibody fragments which bind certain proteins,such as the proteins of a second cellular extract. The bacteriophagewhich are eliminated are removed from the library by the bindingaffinity of their displayed antibody fragments to the certain proteins.

For instance, a method of preparing an antibody array optionally beginswith a phage display library prepared from RNA isolated from the spleensof mice previously immunized with a lysate of human T-cells. The phagelibrary is then passed over a column or affinity surface comprisingproteins from the lysates of background cells such as human fibroblastswhich have been cross-linked to a surface or resin. The phage remainingin the flowthrough solution from the first column/affinity surface isthen passed over a second affinity surface, such as a chromatographycolumn, containing cross-linked proteins prepared from a lysate of humanT-cells. The flowthrough solution from the second column/affinitysurface is then discarded since this solution contains phage whichdisplays recombinant antibodies that did not react with the secondaffinity surface. Phage which specifically react with the secondaffinity surface and remain bound to the second affinity surface arethen collected by elution. Elution can be achieved by lowered pH (2.0),increased ionic strength, or proteolytic release by a specificproteolytic cut site genetically engineered between the displayedrecombinant antibody and the gene III protein of the phage. In a nextstep of the method, the eluted phage are separated into isolated plaquesby plating and then propagated as separate cultures. Periplasmicfractions from the separate cultures are prepared and the correspondingrecombinant antibodies purified. The purified recombinant antibodies arethen dispensed into separate patches on a 2-D array where they areimmobilized onto an organic thinfilm.

Methods of preparing an array of protein-capture agents where theprotein-capture agents have been selected against the proteins of acellular extract, or a body fluid, create arrays of protein-captureagents where all of the binding partners of the arrays are not initiallyknown. The primary information provided by binding of proteins to thesetypes of arrays is contained in the pattern of protein abundance. Onceinteresting patches on an array have been identified by comparison ofthe protein expression pattern to that of a control (for instance, itmay be observed that there is a significant increase in the amount ofprotein bound to a patch of the array following exposure of a cell to acertain set of conditions), the identity of the protein ligand bindingto a particular patch on the array can be assessed by affinitypurification of the protein ligand followed by microsequencing and/ormass spectrometry or the like.

An alternative method for producing an array of protein-capture agentscomprises: selecting protein-capture agents from a library ofprotein-capture agents, wherein the protein-capture agents are selectedby their binding affinity to proteins expressed by a cDNA expressionlibrary; producing a plurality of purified samples of the selectedprotein-capture agents; and immobilizing each different purifiedprotein-capture agent onto an organic thinfilm on a separate patch onthe surface of a substrate to form a plurality of patches on discrete,known regions of the substrate surface covered by organic thinfilm

This method also optionally comprises the additional step of biasing theprotein-capture agent library by eliminating from the library thoseprotein-capture agents which bind certain proteins, such as the proteinsof a cellular extract, wherein the protein-capture agents which areeliminated are removed from the library by their binding affinity tosaid certain proteins. In most cases, the proteins which are used tosubtract protein-capture agents from the library of protein-captureagents would be immobilized. This step of biasing the library mayoptionally occur after the selection step by affinity binding to theproteins expressed by the cDNA expression library, but more typically,it occurs prior to that selection step. The order of these step willdepend on the nature of the selection and elution steps. One of ordinaryskill in the art will readily be able to determine an appropriate seriesof steps. In the optional step of biasing the library of protein-captureagents, the library is optionally biased to eliminate protein-captureagents that recognize common proteins or proteins of non-interest (asdescribed above for a previous embodiment). Preferably, the methodfurther comprises the additional step of identifying which individualselected protein-capture agents bind which individual proteins expressedby the cDNA expression library.

In another preferred embodiment of the the method, the protein-captureagents are antibody fragments displayed on the surface of recombinantbacteriophages and the library of protein-capture agents is a phagedisplay library.

For instance, one example of a method of preparing an array ofantibodies optionally begins with a phage display library prepared fromRNA isolated from the spleens of mice previously immunized with a lysateof human T-cells. The phage library is then passed over a column oraffinity surface comprising proteins from the lysates of backgroundcells such as human fibroblasts which have been cross-linked to asurface or resin. The phage remaining in the flowthrough solution fromthe first column/affinity surface is then collected. A cDNA expressionlibrary derived from message RNA (mRNA) isolated from human T-cells isprepared in which the expressed proteins from the expression library aregenetically fused with an expression tag (such as a six histidine tag).The library is expanded and the tagged proteins are collectivelyexpressed and purified. The pool of purified, tagged proteins from thecDNA expression library is cross-linked to an affinity surface, such asa chromatography column. The phage display library which passed throughthe first affinity surface or column is passed over the affinity surfacebearing the immobilized proteins of the cDNA expression library. Theflowthrough solution containing phage displaying recombinant antibodiesthat did not react with the affinity surface is discarded. Phage whichspecifically react with the affinity surface are collected by elutionachieved by lowering the pH (2.0). Cells from the cDNA expressionlibrary are plated and a filter lift of the colonies is made usingnitrocellulose or charged nylon filters. Reactive sites on the filterare blocked with a standard blocking solution and the filters are probedwith the selected bacteriophage eluted off of the second column. Thephage are visualized by reaction with a monoclonal antibody recognizingthe gene VIII coat protein of the bacteriophage, conjugated to alkalinephosphatase. Reactive sites on the filter are cut out and the phageeluted from the filter pieces and propagated separately. The elutedphage are separated into isolated plaques and then propagated asseparate cultures. Periplasmic fractions from the separate cultures areprepared and the corresponding recombinant antibodies purified. Thepurified recombinant antibodies are then dispensed onto separate patchesof organic thinfilm on a 2-D array. Samples are reacted with the arrayand protein ligands with interesting differential abundance patterns(when compared to a control) are identified. Colonies on the originalplate corresponding to the phage-reactive sites on the filter arepropagated and the plasmids containing the cDNA sequenced to identifythe protein ligands reactive with the recombinant antibodies of thephage.

In the preparation of the arrays of the invention, phage display methodsanalogous to those used for antibody fragments may be used forprotein-capture agents other than antibody fragments as long as theprotein-capture agent is composed of protein and is of suitable size tobe incorporated into the phagemid or alternative vector and expressed asa fusion with a bacteriophage coat protein. Phage display techniquesusing non-antibody libraries typically make use of some type of proteinhost scaffold structure which supports the variable regions. Forinstance, β-sheet proteins, α-helical handle proteins, and other highlyconstrained protein structures have been used as host scaffolds.

Alternative display vectors may also be used to produce theprotein-capture agents, such as antibody moieties, which are printed onthe arrays of the invention. Polysomes, stable protein-ribosome-mRNAcomplexes, can be used to replace live bacteriophage as the displayvehicle for recombinant antibody fragments or other proteins (Hanes andPluckthun, Proc. Natl. Acad. Sci USA, 94:4937-4942, 1997). The polysomesare formed by preventing release of newly synthesized and correctlyfolded protein from the ribosome. Selection of the polysome library isbased on binding of the antibody fragments or other proteins which aredisplayed on the polysomes to the target protein. mRNA which encodes thedisplayed protein or antibody having the desired affinity for the targetis then isolated. Larger libraries may be used with polysome displaythan with phage display.

In still another alternative method of preparing the protein-captureagents of the arrays of the invention, an alternative display method ofselection such as lambda display (Mikawa et al., J. Mol. Biol.,262:21-30,1 996), bacterial display (Georgiou et al., Nat. Biotechnol.,15:29-34, 1997) or eukaryotic cell display may instead by used.

Furthermore, selection methods other than display methods may also beused in the preparation of protein-capture agents for the arrays of theinvention. As indicated above, the protein-capture agents may beobtained by any in vitro or in vivo selection procedure known to thoseskilled in the art. In one embodiment of the invention, protein-captureagents other than antibodies and antibody fragments are batch selectedon the protein in cellular extracts. Such procedures generate adiversity of protein-capture agents which are highly suitable forapplications in proteomics.

In alternative embodiments of the invention, the protein-capture agentsare partially or wholly prepared by synthetic means. If theprotein-capture agent is a protein, then methods of peptide synthetic orprotein ligation may optionally be used to construct a protein fromamino acid or polypeptide building blocks. Protein-capture agents whichare polynucleotides are readily prepared synthetically.

(g) Uses of the Arrays

The present invention also provides methods of using the inventionarrays. In general, for a variety of applications including proteomicsand diagnostics, the methods of the invention involve the delivery ofthe sample containing the proteins to be analyzed to the arrays. Afterthe proteins of the sample have been allowed to interact with and becomeimmobilized on the patches of the array comprising protein captureagents with the appropriate biological specificity, the presence and/oramount of protein bound at each patch is then determined.

Use of one of the protein-capture agent arrays of the invention mayoptionally involve placing the two-dimensional array in a flowchamberwith approximately 1-10 microliters of fluid volume per 25 mm² overallsurface area. The cover over the array in the flowchamber is preferablytransparent or translucent. In one embodiment, the cover may comprisePyrex or quartz glass. In other embodiments, the cover may be part of adetection system that monitors interaction between the protein-captureagents immobilized on the array and protein in a solution such as acellular extract. The flowchambers should remain filled with appropriateaqueous solutions to preserve protein activity. Salt, temperature, andother conditions are preferably kept similar to those of normalphysiological conditions. Proteins in a fluid solution may be flushedinto the flow chamber as desired and their interaction with theimmobilized protein-capture agents determined. Sufficient time must begiven to allow for binding between the protein-capture agent and itsbinding partner to occur. The amount of time required for this will varydepending upon the nature and tightness of the affinity of theprotein-capture agent for its binding partner. No specializedmicrofluidic pumps, valves, or mixing techniques are required for fluiddelivery to the array.

Alternatively, protein-containing fluid can be delivered to each of thepatches of the array individually. For instance, in one embodiment, theregions of the substrate surface may be microfabricated in such a way asto allow integration of the array with a number of fluid deliverychannels oriented perpendicular to the array surface, each one of thedelivery channels terminating at the site of an individualprotein-capture agent-coated patch.

The sample which is delivered to the array will typically be a fluid. Ina preferred embodiment of the invention, the sample is a cellularextract or a body fluid. The sample to be assayed may optionallycomprise a complex mixture of proteins, including a multitude ofproteins which are not binding partners of the protein-capture agents ofthe array. If the proteins to be analyzed in the sample are membraneproteins, then those proteins will typically need to be solubilizedprior to administration of the sample to the array. If the proteins tobe assayed in the sample are proteins secreted by a population of cellsin an organism, a sample which is derived from a body fluid ispreferred. If the proteins to be assayed in the sample areintracellular, a sample which is a cellular extract is preferred. In oneembodiment of the invention, the array may comprise protein-captureagents which bind fragments of the expression products of a cell orpopulation of cells in an organism. In such a case, the proteins in thesample to be assayed may have been prepared by performing a digest ofthe protein in a cellular extract or a body fluid. In an alternativeapplication of the array, the proteins from only specific fractions of acell are collected for analysis in the sample.

In general, delivery of solutions containing proteins to be bound by theprotein-capture agents of the array may optionally be preceded,followed, or accompanied by delivery of a blocking solution. A blockingsolution contains protein or another moiety which will adhere to sitesof non-specific binding on the array. For instance, solutions of bovineserum albumin or milk may be used as blocking solutions.

It is understood that some proteins a sample which are not the intendedbinding partner of the protein-capture agents of a patch (and may, infact, be the intended binding partner of another patch) on the array maystill bind to the patch to some degree. Preferably, this type of bindingonly occurs to a very minor degree. Also, it is understood that evenwhen the correct binding partners are present in the solution beingassayed, the binding partners will bind to the patch comprising theirprotein-capture agent with less than 100% efficiency.

A wide range of detection methods is applicable to the methods of theinvention. As desired, detection may be either quantitative orqualitative. The invention array can be interfaced with opticaldetection methods such as absorption in the visible or infrared range,chemoluminescence, and fluorescence (including lifetime, polarization,fluorescence correlation spectroscopy (FCS), and fluorescence-resonanceenergy transfer (FRET)). Furthermore, other modes of detection such asthose based on optical waveguides PCT Publication (WO 96/26432 and U.S.Pat. No. 5,677,196), surface plasmon resonance, surface charge sensors,and surface force sensors are compatible with many embodiments of theinvention. Alternatively, technologies such as those based on BrewsterAngle microscopy (BAM) (Schaaf et al., Langmuir, 3:1131-1135 (1987)) andellipsometry (U.S. Pat. Nos. 5,141,311 and 5,116,121; Kim,Macromolecules, 22:2682-2685 (1984)) could be applied. Quartz crystalmicrobalances and desorption processes (see for example, U.S. Pat. No.5,719,060) provide still other alternative detection means suitable forat least some embodiments of the invention array. An example of anoptical biosensor system compatible both with some arrays of the presentinvention and a variety of non-label detection principles includingsurface plasmon resonance, total internal reflection fluorescence(TIRF), Brewster Angle microscopy, optical waveguide lightmodespectroscopy (OWLS), surface charge measurements, and ellipsometry canbe found in U.S. Pat. No. 5,313,264.

Although non-label detection methods are generally preferred, some ofthe types of detection methods commonly used for traditionalimmunoassays which require the use of labels may be applied to thearrays of the present invention. These techniques include noncompetitiveimmunoassays, competitive immunoassays, and dual label, ratiometricimmunoassays. These particular techniques are primarily suitable for usewith the arrays of protein-capture agents when the number of differentprotein-capture agents with different specificity is small (less thanabout 100). In the competitive method, binding-site occupancy isdetermined indirectly. In this method, the protein-capture agents of thearray are exposed to a labeled developing agent, which is typically alabeled version of the analyte or an analyte analog. The developingagent competes for the binding sites on the protein-capture agent withthe analyte. The fractional occupancy of the protein-capture agents ondifferent patches can be determined by the binding of the developingagent to the protein-capture agents of the individual patches. In thenoncompetitive method, binding site occupancy is determined directly. Inthis method, the patches of the array are exposed to a labeleddeveloping agent capable of binding to either the bound analyte or theoccupied binding sites on the protein-capture agent. For instance, thedeveloping agent may be a labeled antibody directed against occupiedsites (i.e., a “sandwich assay”). Alternatively, a dual label,ratiometric, approach may be taken where the protein-capture agent islabeled with one label and the second, developing agent is labeled witha second label (Ekins, et al., Clinica Chimica Acta., 194:91-114, 1990).Many different labeling methods may be used in the aforementionedtechniques, including radioisotopic, enzymatic, chemiluminescent, andfluorescent methods. Fluorescent methods are preferred.

FIG. 8 shows a schematic diagram of one type of fluorescence detectionunit which may be used to monitor interaction of immobilizedprotein-capture agents of an array with a protein analyte. In theillustrated detection unit, the array of protein-capture agents 21 ispositioned on a base plate 20. Light from a 100W mercury arc lamp 25 isdirected through an excitation filter 24 and onto a beam splitter 23.The light is then directed through a lens 22, such as a Micro Nikkor 55mm 1:2:8 lens, and onto the array 21. Fluorescence emission from thearray returns through the lens 22 and the beam splitter 23. After nextpassing through an emission filter 26, the emission is received by acooled CCD camera 27, such as the Slowscan TE/CCD-1024SF&SB (PrincetonInstruments). The camera is operably connected to a CPU 28 which is inturn operably connected to a VCR 29 and a monitor 30.

FIG. 9 shows a schematic diagram of an alternative detection methodbased on ellipsometry. Ellipsometry allows for information about thesample to be determined from the observed change in the polarizationstate of a reflected light wave. Interaction of a protein analyte with alayer of immobilized protein-capture agents on a patch results in athickness change and alters the polarization status of a plane-polarizedlight beam reflected off the surface. This process can be monitored insitu from aqueous phase and, if desired, in imaging mode. In a typicalsetup, monochromatic light (e.g. from a He—Ne laser, 30) is planepolarized (polarizer 31) and directed onto the surface of the sample anddetected by a detector 35. A compensator 32 changes the ellipticallypolarized reflected beam to plane-polarized. The corresponding angle isdetermined by an analyzer 33 and then translated into the ellipsometricparameters Psi and Delta which change upon binding of protein with theprotein-capture agents. Additional information can be found in Azzam, etal., Ellipsometry and Polarized Light, North-Holland Publishing Company:Amsterdam, 1977.

The arrays of the present invention are particularly useful forproteomics. Those arrays which comprise significant numbers ofprotein-capture agents of different specificity on separate patches canbind significant numbers of proteins which are expression products, orfragments thereof, of a cell or population of cells in an organism andare particularly suitable for use in applications involving proteomics.For instance, an array with at least about 10³ and up to about 10⁵different protein-capture agents such as antibodies or antibodyfragments can provide a highly comprehensive picture of the proteincontent of the cell under a specific set of conditions.

In one embodiment of the invention, a method of assaying in parallel fora plurality of different proteins in a sample which are expressionproducts, or fragments thereof, of a cell or a population of cells in anorganism, is provided which comprises the following steps: first,delivering the sample to an array of spatially distinct patches ofdifferent protein-capture agents under conditions suitable for proteinbinding, wherein each of the proteins being assayed is a binding partnerof the protein-capture agent of at least one patch on the array; next,optionally washing said array to remove unbound or nonspecifically boundcomponents of the sample from the array; and in a final step, detecting,either directly or indirectly, for the presence or amount of proteinbound to each patch of the array.

In another embodiment of the invention, a method of assaying in parallelfor a plurality of different proteins in a sample which are expressionproducts, or fragments thereof, of a cell or a population of cells in anorganism, comprises first delivering the sample to the invention arrayof protein-capture agents under conditions suitable for protein binding,wherein each of the proteins being assayed is a binding partner of theprotein-capture agent of at least one patch on the array. The first stepmay be followed by an optional step of washing the array with fluid toremove unbound or nonspecifically bound components of the sample fromthe array. Lastly, the presence or amount of protein bound to each patchis detected, either directly or indirectly.

A variety of different embodiments of the invention array ofprotein-capture agents may be used in the methods for assaying inparallel for a plurality of different proteins in a sample which areexpression products, or fragments thereof, of a cell or a population ofcells in an organism. Generally, preferred embodiments of these methodscomprise the use of preferred arrays of the invention. For instance, inpreferred embodiments of the method, the protein-capture agents areantibodies or antibody fragments. In further preferred embodiments forassaying the different amounts of a plurality of proteins in a cell inparallel or the protein expression pattern of a cell, the plurality ofpatches on the array can bind at least about 100 or at least about 10³different proteins which are the expression products, or fragmentsthereof, of a cell or population of cells in an organism. Alternatively,the plurality of patches on the array used in the methods can bind atleast about 10⁴ different proteins which are the expression products, orfragments thereof, of a cell or population of cells in an organism.

The methods of assaying in parallel for a plurality of differentproteins in a sample which are expression products, or fragmentsthereof, of a cell or a population of cells in an organism, optionallycomprise the additional step of further characterizing the protein boundto at least one patch of the array. This step is typically designed toidentify the nature of the protein bound to the protein-capture agent ofa particular patch. In some cases, the entire identity of the boundprotein may not be known and the purpose of the further characterizationmay be the initial indentification of the mass, sequence, structureand/or activity of the bound protein. In other cases, the basic identityof the protein may be known, but the post-translational modification,activation state, or some other feature of the protein may not be known.In one embodiment, the step of further characterizing the proteinsinvolves measuring the activity of the proteins. Although in some casesit may be preferable to remove the protein from the patch before thestep of further characterizing the protein is carried out, in othercases the protein can be further characterized while still bound to thepatch. In still further embodiments, the protein-capture agents of thepatch which binds a protein can be used to isolate and/or purify theprotein from cells. The purified sample can then be characterizedthrough traditional means such as microsequencing, mass spectrometry,and the like.

In another embodiment, the present invention provides a method ofdetermining the protein expression pattern of a cell or population ofcells in an organism. This method involves first delivering a samplecontaining expression products, or fragments thereof, of the cell orpopulation of cells to the protein-capture agent array of the inventionunder conditions suitable for protein binding. The presence and/oramount of protein bound to each patch can then be determined by asuitable detection means. The detection may be either direct orindirect. Quantitative detection is typically preferred for thisapplication (and for other proteomics applications). The methodpreferably further comprises an additional step before the detectionstep comprising washing the array to remove unbound or nonspecificallybound components of the sample from the array. The amount of proteinbound to a patch of the array may optionally be determined relative tothe amount of a second protein bound to a second patch of the array. Themethod of determining the protein expression pattern of a cell or apopulation of cells in an organism, optionally comprises the additionalstep of further characterizing the proteins bound to at least one patchof the array, as previously described above.

In the method of assaying the protein expression pattern of a cell orpopulation of cells in an organism, many of the targets of theprotein-capture agents of the array may optionally be of unknownsequence, identity, and/or function. For instance, the antibodies of thearray may have been prepared by selecting a phage display library byaffinity binding to the immobilized proteins of a cellular extract whichcontains many unidentified proteins. If the protein bound by aprotein-capture agent on a particular patch of an array is unknown, butis of interest, then that protein may optionally be later identified orcharacterized by first using the same protein-capture agent that wasused on the array to isolate the protein in question from cells. Theisolated binding partner from the cell can then be assayed directly forfunction and/or sequenced.

The arrays of protein-capture agents may also be used to compare theprotein expression patterns of two cells or populations of cells. Inthis method, a sample containing expression products, or fragmentsthereof, of a first cell or population of cells is delivered to theinvention array of protein-capture agents under conditions suitable forprotein binding. In an analogous manner, a sample containing expressionproducts, or fragments therof, of a second cell or population of cellsto a second array, is delivered to a second array which is identical tothe first array. Preferably, both arrays are then washed to removeunbound or nonspecifically bound components of the sample from thearrays. In a final step, the amounts of protein remaining bound to thepatches of the first array are compared to the amounts of proteinremaining bound to the corresponding patches of the second array. If itis desired to determine the differential protein expression pattern oftwo cells or populations of cells, for instance, then the amount ofprotein bound to the patches of the first array may be subtracted fromthe amount of protein bound to the corresponding patches of the secondarray.

Methods of comparing the protein expression of two cells or populationsof cells are particularly useful for the understanding of biologicalprocesses. For instance, using these methods, the protein expressionpatterns of identical cells or closely related cells exposed todifferent conditions can be compared. Most typically, the proteincontent of one cell or population of cells is compared to the proteincontent of a control cell or population of cells. For instance, in oneembodiment of the invention, one of the cells or populations of cells isneoplastic and the other cell is not. In another embodiment, one of thetwo cells or populations of cells being assayed is infected with apathogen. Alternatively, one of the two cells or populations of cellshas been exposed to a stressor and the other cell or population of cellsserves as a control. The stressor may optionally be chemical,environmental, or thermal. One of the two cells may optionally beexposed to a drug or a potential drug and its protein expression patterncompared to a control cell.

Such methods of assaying differential gene expression at the proteinlevel are useful in the identification and validation of new potentialdrug targets as well as for drug screening. For instance, the method maybe used to identify a protein which is overexpressed in tumor cells, butnot in normal cells. This protein may be a target for drug intervention.Inhibitors to the action of the overexpressed protein can then bedeveloped. Alternatively, antisense strategies to inhibit theoverexpression may be developed. In another instance, the proteinexpression pattern of a cell, or population of cells, which has beenexposed to a drug or potential drug can be compared to that of a cell,or population of cells, which has not been exposed to the drug. Thiscomparison will provide insight as to whether or not the drug has hadthe desired effect on a target protein (drug efficacy) and whether otherproteins of the cell, or population of cells, have also been affected(drug specificity).

The arrays of the present invention are also suitable for diagnosticapplications and suitable for use in diagnostic devices. The highdensity of the antibodies on some arrays of the present inventionenables a large number of different, antibody-based diagnostic tests tobe formatted onto a single biochip. The protein-capture agents on theinvention array can be used to evaluate the status of a diseasecondition in a tissue, such as a tumor, where the expression levels ofcertain proteins in the cells of the tissue is known to be indicative ofa particular type of disease condition or stage of a disease condition.If certain patterns of protein expression are not previously known to beindicative of a disease state, the protein-capture agent arrays of theinvention can then first be used to establish this information.

Accordingly, in one embodiment, the invention provides a method ofevaluating a disease condition in a tissue of an organism comprisingfirst contacting the invention array of protein-capture agents with asample comprising the expression products, or fragments thereof, of thecells of the tissue being evaluated, wherein the contacting occurs underconditions suitable for protein binding and wherein the binding partnersof a plurality of protein-capture agents on the array include proteinswhich are expression products, or fragment thereof, of the cells of thetissue and whose expression levels are indicative of the diseasecondition. The method next comprises detecting, either directly orindirectly, for the presence of protein to each patch. In a preferredembodiment, the method further comprises the step of washing the arrayto remove unbound or nonspecifically bound components of the sample fromthe array. In such a method, the array will typically compriseprotein-capture agents which bind those proteins whose presence,absence, or relative amount in cells is known to be indicative of aparticular type of disease condition or state of a disease condition.For instance, the plurality of proteins being assayed in such a methodmay include such proteins as HER2 protein or prostate-specific antigen(PSA).

(h) EXAMPLES

The following specific examples are intended to illustrate the inventionand should not be construed as limiting the scope of the claims:

Example 1 Fabrication of a Two-Dimensional Array by Photolithography

In a preferred embodiment of the invention, two-dimensional arrays arefabricated onto the substrate material via standard photolithographyand/or thin film deposition. Alternative techniques include microcontactprinting. Usually, a computer-aided design pattern is transferred to aphotomask using standard techniques, which is then used to transfer thepattern onto a silicon wafer coated with photoresist.

In a typical example, the array (“chip”) with lateral dimensions of10×10 mm comprises squared patches of a bioreactive layer (here: gold asthe coating on a silicon substrate) each 0.1×0.1 mm in size andseparated by hydrophobic surface areas with a 0.2 mm spacing. 4″diameter Si(100) wafers (Virginia Semiconductor) are used as bulkmaterials. Si(100) wafers are first cleaned in a 3:1 mixture of H₂SO₄,conc.: 30% H₂O₂ (90° C., 10 min), rinsed with deionized water (18 MΩcm),finally passivated in 1% aqueous HF, and singed at 150° C. for 30 min tobecome hydrophobic. The wafer is then spincoated with photoresist(Shipley 1813), prebaked for 25 minutes at 90° C., exposed using a KarlSuss contact printer and developed according to standard protocols. Thewafer is then dried and postbaked at 110° C. for 25 min. In the nextstep, the wafer is primed with a titanium layer of 20 nm thickness,followed by a 200 nm thick gold layer. Both layers were deposited usingelectron-beam evaporation (5 Å/s). After resist stripping and a shortplasma treatment, the gold patches can be further chemically modified toachieve the desired bioreactive and biocompatible properties (seeExample 3, below).

Example 2 Fabrication of a Two-dimensional Array by Deposition Through aHole Mask

In another preferred embodiment the array of gold patches is fabricatedby thin film deposition through a hole mask which is in direct contactwith the substrate. In a typical example, Si(100) wafers are firstcleaned in a 3:1 mixture of H₂SO₄, conc.: 30% H₂O₂ (90° C., 10 min),rinsed with deionized water (18 MΩcm), finally passivated in 1% aqueousHF and singed at 150° C. for 30 min to become hydrophobic. The wafer isthen brought into contact with a hole mask exhibiting the positivepattern of the desired patch array. In the next step, the wafer isprimed with a titanium layer of 20 nm thickness, followed by a 200 nmthick gold layer. Both layers were deposited using electron-beamevaporation (5 Å/s). After removal of the mask, the gold patches can befurther chemically modified to achieve the desired bioreactive andbiocompatible properties (see Example 3, below).

Example 3 Synthesis of an Aminoreactive Monolayer Molecule (Followingthe Procedure Outlined in Wagner et al., Biophys. J., 1996,70:2052-2066)

General. ¹H- and ¹³C-NMR spectra are recorded on Bruker instruments (100to 400 MHz). Chemical shifts (δ) are reported in ppm relative tointernal standard ((CH₃)₄Si, δ=0.00 (¹H- and ¹³C-NMR)). FAB-mass spectraare recorded on a VG-SABSEQ instrument (Cs⁺, 20 keV). Transmissioninfrared spectra are obtained as dispersions in KBr on an FTIRPerkin-Elmer 1600 Series instrument. Thin-layer chromatography (TLC) isperformed on precoated silica gel 60 F254 plates (MERCK, Darmstadt,FRG), and detection was done using Cl₂/toluidine, PdCl₂ and UV-detectionunder NH₃-vapor. Medium pressure liquid chromatography (MPLC) isperformed on a Labomatic MD-80 (LABOMATIC INSTR. AG, Allschwil,Switzerland) using a Buechi column (460×36 mm; BUECHI, Flawil,Switzerland), filled with silica gel 60 (particle size 15-40 μm) fromMerck.

Synthesis of 11,11′-dithiobis(succinimidylundecanoate) (DSU). Sodiumthiosulfate (55.3 g, 350 mmol) is added to a suspension of11-bromo-undecanoic acid (92.8 g, 350 mmol) in 50% aqueous 1,4-dioxane(1000 ml). The mixture is heated at reflux (90° C.) for 2 h until thereaction to the intermediate Bunte salt was complete (clear solution).The oxidation to the corresponding disulfide is carried out in situ byadding iodine in portions until the solution retained with a yellow tobrown colour. The surplus of iodine is retitrated with 15% sodiumpyrosulfite in water. After removal of 1,4-dioxane by rotary evaporationthe creamy suspension is filtered to yield product11,11′-dithiobis(undecanoic acid). Recrystallization from ethylacetate/THF provides a white solid (73.4 g, 96.5%): mp 94° C.; ¹H NMR(400 MHz, CDCl₃/CD₃OD 95:5): δ 2.69 (t, 2H, J=7.3 Hz), 2.29 (t, 2H,J=7.5 Hz), 1.76-1.57 (m, 4H), and 1.40-1.29 (m, 12H); FAB-MS (Cs⁺, 20keV): m/z (relative intensity) 434 (100, M⁺). Anal. Calcd. forC₂₂H₄₂O₄S₂: C, 60.79; H, 9.74; S, 14.75. Found: C, 60.95; H, 9.82; S,14.74. To a solution of 11,11′-dithiobis(undecanoic acid) (1.0 g, 2.3mmol) in THF (50 ml) is added N-hydroxysuccinimide (0.575 g, 5 mmol)followed by DCC (1.03 g, 5 mmol) at 0° C. After the reaction mixture isallowed to warm to 23° C. and is stirred for 36 h at room temperature,the dicyclohexylurea (DCU) is filtered. Removal of the solvent underreduced pressure and recrystallization from acetone/hexane provides11,11′-dithiobis(succinimidylundecanoate) as a white solid. Finalpurification is achieved by medium pressure liquid chromatography (9bar) using silica gel and a 2:1 mixture of ethyl acetate and hexane. Theorganic phase is concentrated and dried in vacuum to afford11,11′-dithiobis(succinimidylundecanoate) (1.12 g, 78%): mp 95° C.; ¹HNMR (400 MHz, CDCl₃): δ 2.83 (s, 4H), 2.68 (t, 2H, J=7.3 Hz), 2.60 (t,2H, J=7.5 Hz), 1.78-1.63 (m, 4H), and 1.43-1.29 (m, 12H); FAB-MS (Cs⁺,20 keV): m/z (relative intensity) 514 (100), 628 (86, M⁺). Anal. Calcd.for C₃₀H₄₈N₂O₈S₂: C, 57.30; H, 7.69; N, 4.45; S, 10.20. Found: C, 57.32;H, 7.60; N, 4.39; S, 10.25.

Example 4 Formation of an Aminoreactive Monolayer on Gold (Following theProcedure of Wagner et al., Biophys. J., 1996, 70:2052-2066)

Monolayers based on 11,11′-dithiobis(succinimidylundecanoate) (DSU) canbe deposited on Au(111) surfaces of substrates described under Examples1 and 2 by immersing them into a 1 mM solution of DSU in chloroform atroom temperature for 1 hour. After rinsing with 10 volumes of solvent,the N-hydroxysuccinimidyl-terminated monolayer is dried under a streamof nitrogen and immediately used for immobilization of theprotein-capture agents.

Example 5 Formation and Use of an Array of Immobilized Fab′ AntibodyFragments to Detect Concentrations of Soluble Proteins Prepared fromCultured Mammalian Cells

Collections of IgG antibodies are purchased from commercial sources(e.g. Pierce, Rockford, Ill.). The antibodies are first purified byaffinity chromatography based on binding to immobilized protein A. Theantibodies are diluted 1:1 in binding buffer(0.1 M Tris-HCl, 0.15 MNaCl, pH 7.5). A 2 ml minicolumn containing a gel with immobilizedprotein A is prepared. (Hermanson, et. al., Immobilized Affinity LigandTechniques, Academic Press, San Diego, 1992.) The column is equilibratedwith 10 ml of binding buffer. Less than 10 mg of immunoglobulin isapplied to each 2 ml minicolumn and the column is washed with bindingbuffer until the absorbance at 280 nm is less than 0.02. The boundimmunoglobulins are eluted with 0.1 M glycine, 0.15 M NaCl, pH 2.8, andimmediately neutralized with 1.0 M Tris-HCl, pH 8.0 to 50 mM finalconcentration and then dialyzed against 10 mM sodium phosphate, 0.15 MNaCl, pH 7.2 and stored at 4° C.

The purified immunoglobulin are digested with immobilized pepsin. Pepsinis an acidic endopeptidase and hydrolyzes proteins favorably adjacent toaromatic and dicarboxylic L-amino acid residues. Digestion of IgG withpepsin generates intact F(ab′)₂ fragments. Immobilized pepsin gel iswashed with digestion buffer; 20 mM sodium acetate, pH 4.5. A solutionof purified IgG at 10 mg/ml is added to the immobilized pepsin gel andincubated at 37° C. for 2 hours. The reaction is neutralized by theaddition of 10 mM Tris-HCl, pH 7.5 and centrifuged to pellet the gel.The supernatant liquid is collected and applied to an immobilizedprotein A column, as described above, to separate the F(ab′)₂ fragmentsfrom the Fc and undigested IgG. The pooled F(ab′)₂ is dialyzed against10 mM sodium phosphate, 0.15 M NaCl, pH 7.2 and stored at 4° C. Thequantity of pooled, eluted F(ab′)₂ is measured by peak area absorbanceat 280 nm.

The purified F(ab′)₂ fragments at a concentration of 10 mg/ml arereduced at 37° C. for 1 hour in a buffer of 10 mM sodium phosphate, 0.15M NaCl, 10 mM 2-mercaptoethylamine, 5 mM EDTA, pH 6.0. The Fab′fragments are separated from unsplit F(ab′)₂ fragments and concentratedby application to a Sephadex G-25 column (M_(r)=46,000-58,000). Thepooled Fab′ fragments are dialyzed against 10 mM sodium phosphate, 0.15M NaCl, pH 7.2. The reduced Fab′ fragments are diluted to 100 μg/ml andapplied onto the bioreactive patches containing exposed aminoreactivefunctional groups using a computer-aided, capillary-basedmicrodispensing system (for antibody immobilization procedures, seeDammer et al., Biophys. J., 70:2437-2441, 1996). After an immobilizationperiod of 30 minutes at 30° C., the array is rinsed extensively with 10mM sodium phosphate, 0.15 M NaCl, 5 mM EDTA, pH 7.0.

Transformed human cells grown in culture are collected by low speedcentrifugation, briefly washed with ice-cold phosphate-buffered solution(PBS), and then resuspended in ice-cold hypotonic buffer containingDNase/RNase (10 μg/ml each, final concentration) and a mixture ofprotease inhibitors. Cells are transferred to a microcentrifuge tube,allowed to swell for 5 minutes, and lysed by rapid freezing in liquidnitrogen and thawing in ice-cold water. Cell debris and precipitates areremoved by high-speed centrifugation and the supernatant is cleared bypassage through a 0.45 μm filter. The cleared lysate is applied to theFab′ fragment array described above and allowed to incubate for 2 hoursat 30° C. After binding the array is washed extensively with 10 mMsodium phosphate, 0.15 M NaCl, 5 mM EDTA, pH 7.0. The location andamount of bound proteins are determined by optical detection.

Example 6 Formation and Use of an Array of Immobilized AntibodyFragments to Detect Concentrations of Soluble Proteins Prepared fromCultured Mammalian Cells

A combinatorial library of filamentous phage expressing scFv antibodyfragments is generated based on the technique of McCafferty andcoworkers; McCafferty, et al., Nature, 1990, 348:552-554; Winter andMilstein, Nature, 1991, 349:293-299. Briefly, mRNA is purified frommouse spleens and used to construct a cDNA library. PCR fragmentsencoding sequences of the variable heavy and light chain immunoglobulingenes of the mouse are amplified from the prepared cDNA. The amplifiedPCR products are joined by a linker region of DNA encoding the 15 aminoacid peptide (Gly₄SerGly₂CysGlySerGly₄Ser) (SEQ ID NO: 1) and theresulting full-length PCR fragment is cloned into an expression plasmid(pCANTAB 5 E) in which the purification peptide tag (E Tag) has beenreplaced by a His₆ peptide (SEQ ID NO: 2). Electrocompetent TG1 E.colicells are transformed with the expression plasmid by electroporation.The pCANTAB-transformed cells are induced to produced functionalfilamentous phage expressing scFv fragments by superinfection withM13KO7 helper phage. Cells are grown on glucose-deficient mediumcontaining the antibiotics ampicillin (to select for cells with thephagemid) and kanamycin (to select for cells infected with M13KO7). Inthe absence of glucose, the lac promoter present on the phagemid is nolonger repressed, and synthesis of the scFv-gene 3 fusion begins.

Proteins from a cell lysate are adsorbed to the wells of a 96-wellplate. Transformed human cells grown in culture are collected by lowspeed centrifugation and the cells are briefly washed with ice-cold PBS.The washed cells are then resuspended in ice-cold hypotonic buffercontaining DNase/RNase (10 μg/ml each, final concentration) and amixture of protease inhibitors, allowed to swell for 5 minutes, andlysed by rapid freezing in liquid nitrogen and thawing in ice-coldwater. Cell debris and precipitates are removed by high-speedcentrifugation and the supernatant is cleared by passage through a 0.45μm filter. The cleared lysate is diluted to 10 μg/ml in dilution buffer;20 mM PIPES, 0.15 M NaCl, 0.1% CHAPS, 10%, 5 mM EDTA, 5 mM2-mercaptoethanol, 2 mM DTT, pH 7.2 and applied to the 96-plate wells.After immobilization for 1 hour at 30° C., the well is washed with thedilution buffer and then incubated with dilution buffer containing 10%nonfat dry milk to block unreacted sites. After the blocking step, thewell is washed extensively with the dilution buffer.

Phage expressing displayed antibodies are separated from E. coli cellsby centrifugation and then precipitated from the supernatant by theaddition of 15% w/v PEG 8000, 2.5 M NaCl followed by centrifugation. Thepurified phage are resuspended in the dilution buffer containing 3%nonfat dry milk and applied to the well containing the immobilizedproteins described above, and allowed to bind for 2 hours at 37° C.,followed by extensive washing with the binding buffer. Phage are elutedfrom the well with an elution buffer; 20 mM PIPES, 1 M NaCl, 0.1% CHAPS,10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH 7.2. The well isthen extensively washed with purge buffer; 20 mM PIPES, 2.5 M NaCl, 0.1%CHAPS, 10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH 7.2. Thewell is then extensively washed with dilution buffer; 20 mM PIPES, 0.15M NaCl, 0.1% CHAPS, 10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH7.2. The eluted phage solution is then re-applied to a new wellcontaining adsorbed antigen and the panning enrichment is repeated 4times. Finally, the phage are eluted from the well with 2M of NaCl in 20mM PIPES, 0.1% CHAPS, 10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT,pH 7.2. Eluates are collected and mixed with log-phase TG1 cells, andgrown at 37° C. for 1 hour and then plated onto SOB medium containingampicillin and glucose and allowed to grow for 12-24 hours.

Individual colonies are picked and arrayed into 96-well 2 ml blockscontaining SOB medium and M13KO7 helper phage and grown for 8 hours withshaking at 37° C. The phage are separated from cells by centrifugationand precipitated with PEG/NaCl as described above. Concentrated phageare used to infect HB2151 E. coli. E. coli TG1 produces a suppressortRNA which allows readthrough (suppression) of an amber stop codonlocated between the scFv and phage gene 3 sequences of the pCANTAB 5 Eplasmid. Infected HB2151 cells are selected on medium containingampicillin, glucose, and nalidixic acid. Cells are grown to mid-log andthen centrifuged and resuspended in medium lacking glucose and growthcontinued. Soluble scFv fragments will accumulate in the cell periplasm.A periplasmic extract is prepared from pelleted cells by mild osmoticshock. The soluble scFv released into the supernatant is purified byaffinity binding to Ni-NTA activated agarose and eluted with 10 mM EDTA.

The purified scFv antibody fragments are diluted to 100 μg/ml andapplied onto the bioreactive patches with exposed aminoreactive groupsusing a computer-aided, capillary-based microdispensing system. After animmobilization period of 30 minutes at 30° C., the array is rinsedextensively with 10 mM sodium phosphate, 0.15 M NaCl, 5 mM EDTA, pH 7.0.

Transformed human cells grown in culture are collected by low speedcentrifugation, briefly washed with ice-cold PBS, and then resuspendedin ice-cold hypotonic buffer containing DNase/RNase (10 μg/ml each,final concentration) and mixture of protease inhibitors. Cells aretransferred to a microcentrifuge tube, allowed to swell for 5 minutes,and lysed by rapid freezing in liquid nitrogen and thawing in ice-coldwater. Cell debris and precipitates are removed by high-speedcentrifugation and the supernatant is cleared by passage through a 0.45μm filter. The cleared lysate is applied to the scFv fragment arraydescribed above and allowed to incubate for 2 hours at 30° C. Afterbinding, the array is washed extensively with 0.1 M sodium phosphate,0.15 M NaCl, 5 mM EDTA pH 7.0. The location and amount of bound proteinsare determined by optical detection.

Patterns of binding are established empirically by testing dilutions ofa control cell extract. Extracts from experimental cells are diluted toa series of concentrations and then tested against the array. Patternsof protein expression in the experimental cell lysates are compared toprotein expression patterns in the control samples to identify proteinswith unique expression profiles.

Example 7 Formation and Use of an Array of Immobilized MonoclonalAntibodies to Detect Concentrations of Soluble Proteins Prepared fromCultured Mammalian Cells

Collections of monoclonal antibodies are purchased from commercialsuppliers as either raw ascities fluid or purified by chromatographyover protein A, protein G, or protein L. If from raw ascites fluid, theantibodies are purified using a HiTrap Protein G or HiTrap Protein Acolumn (Pharmacia) as appropriate for the immunoglobulin subclass andspecies. Prior to chromotography the ascites are diluted with an equalvolume of 10 mM sodium phosphate, 0.9% NaCl, pH 7.4 (PBS) and clarifiedby passage through a 0.22 μm filter. The filtrate is loaded onto thecolumn in PBS and the column is washed with two column volumes of PBS.The antibody is eluted with 100 mM Glycine-HCl, pH 2.7 (for protein G)or 100 mM citric acid, pH 3.0 (for protein A). The eluate is collectedinto {fraction (1/10)} volume 1 M Tris-HCl, pH 8.0. The final pH is 7.5.Fractions containing the antibodies are confirmed by SDS-PAGE and thenpooled and dialyzed against PBS.

The different samples of purified antibodies are each diluted to 100μg/ml. Each different antibody sample is applied to a separate patch ofan array of aminoreactive monolayer patches (see Example 4, above) usinga computer-aided, capillary-based microdispensing system. After animmobilization period of 30 minutes at 30° C., the array is rinsedextensively with 10 mM sodium phosphate, 0.15 M NaCl, 5 mM EDTA, pH 7.0.

Transformed human cells grown in culture are collected by low speedcentrifugation, briefly washed with ice-cold PBS, and resuspended inice-cold hypotonic buffer containing Dnase/Rnase (10 μg/ml each, finalconcentration) and a mixture of protease inhibitors. Cells aretransferred to a microcentrifuge tube, allowed to swell for 5 minutes,and lysed by rapid freezing in liquid nitrogen and thawing in ice-coldwater. Cell debris and precipitates are removed by high-speedcentrifugation and the supernatant is cleared by passage through a 0.45μm filter. The cleared lysate is applied to the monoclonal antibodyarray described above and allowed to incubate for 2 hours at 30° C.After binding the array is washed extensively as in Example 6, above.The location and amount of bound proteins are determined by opticaldetection.

All documents cited in the above specification are herein incorporatedby reference. In addition, the copending U.S. patent application “Arraysof Proteins and Methods of Use Thereof”, filed on Jul. 14, 1999, withthe identifier 24406-0004 P1, for the inventors Peter Wagner, DanaAult-Riche, Steffen Nock, and Christian Itin, is herein incorporated byreference in its entirety. Various modifications and variations of thepresent invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in the art are intended tobe within the scope of the following claims.

1. A method for making an array device comprising the steps of: a)providing a substrate having a surface; and one or more proteinimmobilization regions on said surface, said protein immobilizationregions each comprising; i. an ordered hydrophobic monolayer formed ofalkyl chains having proximal ends which are chemisorbed or physisorbedto said surface within said immobilization regions, and oppositehydrophobic distal ends; ii. a hydrophilic monolayer attached to saidordered hydrophobic monolayer, said hydrophilic monolayer comprising aset of first hydrophilic chains, each first hydrophilic chain having aproximal end by which said first hydrophilic chain is linked to an alkylchain distal end, and an opposite hydrophilic distal end; iii. one oremore functional groups, each for covalently attaching a protein captureagent thereto and each covalently attached to one or more of saidhydrophilic distal ends of said first hydrophilic chains; b) reactingone or more of said protein capture agents to one or more of saidfunctional groups attached to a first subset of said set of firsthydrophilic chains within said immobilization regions to attach said oneor more protein capture agents through a residue formed by said reactionof said protein capture agents with said functional groups of said firstsubset of said first hydrophilic chains; and, c) reacting one or moresecond hydrophilic chains with a second subset of said set of firsthydrophilic chains so that one or more of said second hydrophilic chainsattach through one or more residues of one or more of said second subsetof said first hydrophilic chains.
 2. The method of claim 1 wherein atleast one of said second hydrophilic chains is polyethylene glycol. 3.The method of claim 1 wherein at least one of said first hydrophilicchains is oligoethylene glycol.